Gibco Neurobiology Protocols Handbook
Gibco Neurobiology Protocols Handbook
Gibco Neurobiology Protocols Handbook
Introduction..................................................................................................................................4
Cell Analysis..............................................................................................................................65
Immunocytochemistry......................................................................................................................72
Electrophysiology.............................................................................................................................75
Molecular Characterization........................................................................................................77
Transfection................................................................................................................................85
Appendix...................................................................................................................................101
Technical Support...........................................................................................................................108
2
Introduction
Introduction
Overview
It has long been thought that the adult mammalian nervous system was incapable of
regeneration after injury. However, recent advances in our understanding of stem cell
biology and neuroscience have opened up new avenues of research for developing
potential treatments for incurable neurodegenerative diseases and neuronal injuries.
Because stem cells have the capacity to self-renew and generate differentiated cells,
stem cell replacement therapy for central and peripheral nervous system disorders
and injuries strives at repopulating the affected neural tissue with neurons and other
neural cells. One of the main strategies towards this end aims to recapitulate the normal
development of the nervous system by activating the endogenous regenerative capacity
of the neural stem cells or by transplanting neural or embryonic cells.
This chapter defines the key concepts in stem cell biology with respect to the nervous
system, presents an overview of neural development, and summarizes the involvement
of neural cell types in specific neural diseases.
Stem Cells
The classical definition of a stem cell requires that it has the capacity to self-renew and
that it possesses potency. Self-renewal is defined as the ability of the stem cell to go
through multiple cycles of cell division while maintaining its undifferentiated state (i.e.,
to generate daughter cells that are identical to their mother). Potency is the ability of the
stem cell to differentiate into specialized cell types.
Neural Stem Cells Neural stem cells (NSC) are stem cells in the nervous system that can self-renew and
give rise to differentiated progenitor cells to generate lineages of neurons as well as glia,
such as astrocytes and oligodendrocytes. This characteristic is known as multipotency.
NSCs and neural progenitor cells are present throughout development and persist in
the adult nervous system. Multiple classes of NSCs have been identified that differ from
each other in their differentiation abilities, their cytokine responses, and their surface
antigen characteristics.
Stem Cells and Cancer An exciting finding has been the discovery that many cancers may be propagated by a
small number of stem cells present in the tumor mass. This was first described in breast
cancers and subsequently in a variety of solid tumors. Several reports have suggested
that cancer stem cells can be identified in the nervous system as well, and that these
cells bear a remarkable similarity to neural stem cells present in early development.
Likewise, cells resembling glial progenitors have been isolated from some glial tumors
suggesting an intriguing link between developmental and cancer biology
Neural Development
The development of the central nervous system (CNS) is initiated early in the
development by the induction of NSCs and neural progenitor cells; this stage in
development is called neural induction. By studying neural induction and neural
development, we can determine the various factors that stimulate or inhibit the
differentiation of NSCs and the requirements of these NSCs and their offspring for
survival and proper function.
Stages of Neural
Development The nervous system is one of the earliest organ systems that differentiate from the
blastula stage embryo. This differentiation can be mimicked in culture and NSCs can
be derived from human ESC cultures over a period of 2–3 weeks. In vivo, the primitive
neural tube forms by approximately the fourth week of gestation by a process termed
primary neurulation, and neurogenesis commences by the fifth week of development
in humans.
4
Introduction
Like the VZ, the SVZ can be divided into subdomains that express different
rostrocaudal markers and generate phenotypically distinct progeny. Distinct SVZ
domains include the cortical SVZ, the medial ganglion eminence, and the lateral
ganglion eminence. The proportion of SVZ stem cells declines with development and
multipotent stem cells are likely to be present only in regions of ongoing neurogenesis
(e.g., anterior SVZ and the SVZ underlying the hippocampus) in the adult CNS. At this
stage, marker expression is relatively heterogeneous. Other relatively less characterized
stem cells have also been described.
Neural Precursor Cells Neural stem cells do not generate differentiated progeny directly but rather generate
dividing populations of more restricted precursors analogous to the blast cells or
restricted progenitors described in the hematopoietic lineages. These precursors
can divide and self-renew, but they are located in regions distinct from the stem cell
population and can be distinguished from them by the expression of cell surface and
cytoplasmic markers and their ability to differentiate. Several such classes of precursors
have been identified, including neuronal precursors, bi- and tri-potential glial
precursors that generate astrocytes and oligodendrocytes, as well as unipotent astrocyte
or oligodendrocyte precursors. Other precursors such as a neuron-astrocyte precursor
may also exist and the same precursor may have multiple names. Such precursors can
be distinguished from stem cells by their marker expression, ability to differentiate and
time of development.
Summary
The table below lists some of the neurological disorders that have been studied and
modeled in the laboratory and the cell types involved.
Neural Experimental Cell type Growth Progenitor Marker Mature Trans- Reference
disease model factor cell marker plantation
Spinal cord Transplantation Oligo- EGF, bFGF, OPC OLIG1, A2B5, GalC, RIP, O4 Yes Keirstead et
injury of OPC into dendrocyte RA SOX10, NG2 al., 2005
demyelination model
Multiple Demyelinated axons, Oligo- EGF, bFGF, OPC PDGFR, O4, O1, MBP, No Kang et al.,
sclerosis co-cultured with dendrocyte PDGF, RA A2B5, NG2 PLP 2007
rat hippocampal
neurons
Remyelination Oligo- RA, EGF, OPC PDGFR, NG2, O4, O1, MBP, Yes Izrael et al.,
models dendrocyte bFGF, OLIG1/2, PL 2007
Noggin, SOX10
Vitamin C,
Mouse
laminin
Amyotrophic Transplantation of Motoneuron BDNF, GDNF, Motoneuron BF1, HOXB4, NKX6-1, Yes Lee et al.,
lateral motoneuron progeny AA, RA, SHH, Progenitor NKX6-1/6-2, OLIG2, NGN2, 2007
sclerosis into the developing Noggin OLIG1/2 ISL1, ChAT,
and spinal chick embryo VAChT, HB9,
muscular LHX3, HOX
atrophy
in vitro studies only Motoneuron bFGF, RA, Motoneuron OLIG1/2, NKX6-1, No Li et al.,
SHH, BDNF, Progenitor NKX6-1/6-2, OLIG2,NGN2, 2005
GDNF, IGF-1 NGN2 ISL1, ChAT,
VAchT, HB9,
Synapsin
Parkinson's Not applicable DA neuron SHH, FGF8, DA PAX2, PAX5, MAP2, TH, No Perrier et
disease BDNF, AA, precursor LMX, EN1 AADC, VMAT, al., 2004
TGFβ, TGF-3 NURR1, PTX3
in vitro drug DA neuron FGF2 or DA EN1, OTX2, TH, GABA, No Yan et al.,
screening FGF8, SHH, precursor WNT1, PAX2, EN1, AADC 2005
BDNF, GDNF, GBX2
cAMP, AA
Transplantation into DA neuron FGF2, FGF8, DA EN1, PAX2, TH, TUJ-1 Yes Roy et al.,
the neostriata of SHH, BDNF, precursor OTX2 2006
6-hydroxydopamine- GDNF, FBS
lesioned
Parkinsonian rats
Transplantation DA neuron SHH, FGF8, DA PAX2, EN1, TH, EN1, Yes Park et al.,
into the striatum of BDNF, GDNF, precursor NURR1, AADC 2005
hemi-Parkinsonian AA, IGF-1 LMX1B
rats
6
Introduction
Neural Experimental Cell type Growth Progenitor Marker Mature Trans- Reference
disease model factor cell marker plantation
Glial related Astrocyte related Astrocyte Cyclopamine, — — GFAP, S100, No Lee et al.,
diseases disease human GLAST, 2006
astrocyte BDNF, GNDF
medium
CNS/PNS Peripheral and Peripheral Noggin, NGF Neural NCAM, Peripherin, No Brokhman
diseases central nervous sensory precursor TUJ‑1, BRN3, TH, et al., 2008
system neurons neurons SNAIL, TRK-A
dHAND, SOX9
Macular Not applicable Retinal Noggin, Retinal RX, PAX6, RPE-65 No Lambda et
retinal de- pigmented Dickkopf-1, progenitor LHX2, SIX3 al., 2006
generation epithelium IGF-1
References
Trujillo, C.A., Schwindt, T.T., Martins, et al. 2009. Novel perspectives of neural stem cell
differentiation: from neurotransmitters to therapeutics. Cytometry A 75:38–53.
Brokhman, I., Gamarnik-Ziegler, L., Pomp, O., et al. 2008. Peripheral sensory neurons
differentiate from neural precursors derived from human embryonic stem cells.
Differentiation 76:145–155.
Izrael, M., Zhang, P., Kaufman, R., et al. Human oligodendrocytes derived from
embryonic stem cells: Effect of noggin on phenotypic differentiation in vitro and on
myelination in vivo. Mol Cell Neurosci. 34: 310–323.
Kang, S,M,, Cho, M.S., Seo, H. et al. 2007. Efficient induction of oligodendrocytes from
human embryonic stem cells. Stem Cells 25: 419–424.
Keirstead, H.S., Nistor, G., Bernal, G., et al. 2005. Human embryonic stem cell-derived
oligodendrocyte progenitor cell transplants remyelinate and restore locomotion after
spinal cord injury. J Neurosci. 25: 4694–4705.
Lamba, D.A., Karl, M.O., Ware, C.B., and Reh, T.A. 2006. Efficient generation of retinal
progenitor cells from human embryonic stem cells. Proc Natl Acad Sci U S A. 103:12769–
12774.
Lee, H., Shamy, G.A., Elkabetz, Y., et al. 2007. Directed differentiation and
transplantation of human embryonic stem cell-derived motoneurons. Stem Cells
25:1931–1939.
Lee, D.S., Yu, K., Rho, J.Y., et al. 2006. Cyclopamine treatment of human embryonic stem
cells followed by culture in human astrocyte medium promotes differentiation into
nestin- and GFAP-expressing astrocytic lineage. Life Sciences 80:154 –159.
Li, X.J., Du, Z.W., Zarnowska, E.D., et al. 2005. Specification of motoneurons from
human embryonic stem cells. Nat Biotechnol. 23: 215–221.
Molero, A.E., Gokhan, S., Gonzalez, S., et al. 2009. Impairment of developmental stem
cell-mediated striatal neurogenesis and pluripotency genes in a knock-in model of
Huntington’s disease. Proc Natl Acad Sci U S A. 106: 21900-21905.
Park, C.H., Minn, Y.K., Lee, J.Y., et al. 2005. In vitro and in vivo analyses of human
embryonic stem cell-derived dopamine neurons. J Neurochem. 92:1265–1276.
Perrier, A.L., Tabar, V., Barberi, T., et al. 2004. Derivation of midbrain dopamine
neurons from human embryonic stem cells. Proc Natl Acad Sci U S A. 101:12543–12548.
Roy, N.S., Cleren, C., Singh, S.K., et al. 2006. Functional engraftment of human ES cell-
derived dopaminergic neurons enriched by coculture with telomerase-immortalized
midbrain astrocytes. Nat Med. 212:1259–1268
Yan, Y., Yang, D., Zarnowska, E.D., et al. 2005. Directed differentiation of dopaminergic
neuronal subtypes from human embryonic stem cells. Stem Cells. 23:781–790.
8
Neural Cell Culture and Differentiation
Summary
Neural stem cells (NSC) are valuable resources because of their ability to differentiate
into neurons and glial cells with applications in neuroscience and clinical use for
treatment of neurodegenerative disease and neurological disorders. NSC are obtained
by isolation from tissue, or differentiated from pluripotent cells. This chapter
describes methods for expanding human NSC in cell culture and their subsequent
characterization.
Required Materials
Preparing Media
1. Reconstitute bFGF and EGF with 0.1% BSA solution (in KnockOut™ D-MEM/F-12)
at a concentration of 100 μg/mL. You will need 20 μL of each per 100 mL of complete
medium. Freeze unused portions in aliquots.
2. Mix the following components under aseptic conditions. For larger volumes, increase
the component amounts proportionally.
KnockOut™ D-MEM/F-12 1X 97 mL
GlutaMAX™-I Supplement 2 mM 1 mL
You may observe a white precipitate when thawing StemPro® Neural Supplement; this
precipitate will disappear when the supplement is completely thawed or dissolved.
Preparing Matrix
For culture of adherent cultures, use either Fibronectin or CELLstart™ CTS™ to prepare
a matrix for coating your plates.
Note: CELLstart™ CTS™ should not be frozen, vortex or exposed to vigorous agitation
due to potential gel formation.
2. Coat the surface of the culture vessel with the working solution of CELLstart™ CTS™
(14 mL for T-75, 7 mL for T-25, 3.5 mL for 60-mm dish, 2 mL for 35-mm dish).
3. Incubate the culture vessel at 37°C in a humidified atmosphere of 5% CO2 for 1 hour.
4. Remove the vessel from the incubator and store it until use. Remove all CELLstart™ CTS™
solution immediately before use, and fill the vessel with complete StemPro® NSC SFM.
Note: You may coat the plates in advance and store them at 4°C, wrapped tightly with
Parafilm®, for up to 2 weeks. Do not remove CELLstart™ CTS™ solution until just prior
to use. Make sure the plates do not dry out.
10
Neural Cell Culture and Differentiation
4. Add enough working solution to cover the surface of the culture vessel (10 mL for T-75,
2.5 mL for 60-mm dish, 1.5 mL for 35-mm dish).
5. Incubate the culture vessel at 37°C in a humidified atmosphere of 5% CO2 for 1 hour.
6. Remove the vessel from the incubator and store it until use. Remove Fibronectin
solution immediately before use, and fill the vessel with complete StemPro® NSC SFM.
Note: You may store the Fibronectin-treated plates at 4°C, wrapped tightly with
Parafilm®, for up to 2 weeks. Ensure that plates do not dry out. There is no washing
step needed and use the dish directly after aspiration.
Neural stem cell (NSCs) populations can be expanded from frozen stocks and grown in
StemPro® NSC SFM complete medium as adherent cultures, or as suspension cultures.
In either environment, change the spent culture medium every other day. When the
cells in adherent culture reach >90% confluency, they are ready to be passaged. When
the neurospheres in suspension culture become > 3.5 mm in diameter, they are ready to
be passaged. Cells cultured during expansion can be frozen down to create additional
frozen stocks of higher passage number.
2. Transfer vial of frozen NSC from nitrogen tank to water bath. It is important to make
the transfer immediately to prevent crystal formation.
3. Transfer thawed cells into a 15-mL tube and add warmed 1X KnockOut™ D-MEM/F-12
to 10 mL.
4. Spin down the thawed cells by centrifugation at 1,000 rpm for 4 minutes. Aspirate and
discard the supernatant.
5. Resuspend the cells in StemPro® NSC SFM complete medium and plate on a
CELLstart™ CTS™ or Fibronectin-coated plate at high density (1 × 105 cells/cm2).
Note: Viability of thawed cells should be ~80% if they were frozen following the
cryopreservation protocol described here.
Note: The monolayer lifts off from the culture dish within 30 seconds of application of
TrypLE™ Express or StemPro® Accutase®.
3. Gently pipette to loosen monolayer into a single cell suspension. Neutralize the
treatment by adding 4 mL of medium. Do not treat the cells for longer than 3 minutes
after addition of TrypLE™ Express or StemPro® Accutase®.
4. Spin down the cells by centrifugation at 1,200 rpm for 4 minutes. Aspirate and discard
the supernatant.
2. Leave the tube at room temperature and allow the neurosphere to settle to the bottom
of tube. Alternatively, spin down the cells by centrifugation at 500 rpm (200 × g) for
2 minutes.
3. Aspirate the supernatant carefully, and leave the neurospheres in a minimum volume
of medium.
4. Wash the neurospheres with 10 mL D-PBS without Ca2+ and Mg2+, aspirate the D-PBS
supernatant carefully, and leave the neurospheres in a minimum volume of D-PBS.
5. Add 1 mL of TrypLE™ Express to the spheres and gently triturate neurospheres using a
Pasteur pipette to create a single cell suspension.
7. Spin down the cells by centrifugation at 1,200 rpm for 4 minutes. Aspirate and discard
the supernatant.
10. Seed the cells in fresh medium in a suspension dish (a non-coated flask can be used) at a
density of 200,000 cells/mL.
12
Neural Cell Culture and Differentiation
Cryopreserving Neural
Stem Cells 1. Aspirate the medium and wash with D-PBS without Ca2+ and Mg2+.
3. Gently pipette to loosen monolayer into a single cell suspension. Neutralize the
treatment by adding 4 mL of medium. Do not treat the cells for longer than 3 minutes
after addition of TrypLE™ Express.
4. Spin down the cells by centrifugation at 1,200 rpm for 4 minutes. Aspirate the
supernatant.
Note: Freezing medium (2X) can be prepared on the day of use and stored at 4°C until
use.
7. Add a volume of freezing medium equal to the amount of StemPro® NSC SFM
complete medium used to resuspend the cells in a drop-wise manner.
8. Prepare 1 mL aliquots (1 × 106 cells) in cryovials and place the vials in an isopropanol
chamber.
9. Put the isopropanol chamber at -80°C and transfer the vials to liquid nitrogen storage
the next day.
Amplicon Intron
Target Primer Sequence Tm
size size
14
Neural Cell Culture and Differentiation
Summary
Rat neural stem cells (NSCs) serve as a well-established model for investigating
human brain development, disease processes, and treatment strategies for debilitating
central nervous system (CNS) disorders. This protocol describes the in vitro expansion,
passaging, and morphology of rat fetal NSCs in adherent or neurosphere suspension
cultures.
Required Materials
Cells • GIBCO® Rat Fetal Neural Stem Cells (Cat. no. N7744-100) or homogenous cell
preparation from 14−18 days post-coitum rat brain tissue
Media and Reagents • Dulbecco’s Phosphate-Buffered Saline (D-PBS) (Cat. no. 14040)
• Dulbecco’s Phosphate-Buffered Saline (D-PBS) without calcium or magnesium
(Cat. no. 14190)
• StemPro® NSC SFM (Cat. no. A10509-01)
• StemPro® Accutase® Cell Dissociation Reagent (Cat. no. A11105-01)
• CELLstart™ CTS™ (Cat. no. A10142-01)
• Trypan blue (Cat. no. 15250) (included with the Countess®) or the LIVE/DEAD® Cell
Vitality Assay Kit (Cat. no. L34951)
Special Tools • Countess® Automated Cell Counter (Cat. no. C10227) or hemacytometer
Preparing Media
1. Reconstitute bFGF and EGF with 0.1% BSA solution (in KnockOut™ D-MEM/F-12) at
a concentration of 100 μg/mL. You will need 20 μL of each per 100 mL of complete
medium. Freeze unused portions in aliquots.
2. Mix the following components under aseptic conditions. For larger volumes, increase
the component amounts proportionally.
GlutaMAX™-I Supplement 2 mM 1 mL
You may observe a white precipitate when thawing StemPro® Neural Supplement; this
precipitate will disappear when the supplement is completely thawed or dissolved.
1. Dilute CELLstart™ CTS™ 1:100 in D-PBS with calcium and magnesium (e.g., 50 μL of
CELLstart™ CTS™ into 5 mL of D-PBS).
2. Coat the surface of the culture vessel with the working solution of CELLstart™ CTS™
(14 mL for a T-75 flask, 7 mL for a T-25 flask, 3.5 mL for a 60-mm dish, 2 mL for a
35‑mm dish).
3. Incubate the culture vessel at 37°C in a humidified atmosphere of 5% CO2 for 1 hour.
4. Remove the vessel from the incubator and store at 4°C until use. Remove all
CELLstart™ CTS™ solution immediately before use, and fill the vessel with complete
StemPro® NSC SFM.
Note: You may coat the plates in advance and store them at 4°C, wrapped tightly with
Parafilm, for up to 2 weeks. Do not remove CELLstart™ CTS™ solution until just prior to
using the coated plates. Make sure the plates do not dry out.
16
Neural Cell Culture and Differentiation
• For freshly prepared rat fetal NSCs, after rinsing with D-PBS, resuspend in warmed
complete StemPro® NSC SFM at a density of 1 × 107 viable cells/mL.
• For thawed rat fetal NSCs, after determining the viable cell count, resuspend in
warmed complete StemPro® NSC SFM at a cell density of 1 × 107 viable cells/mL.
2. Plate rat fetal NSCs onto CELLstart™ CTS™-coated culture vessels at a density of
5 × 104 cells/cm2. See the following table for recommended seeding densities for
common culture vessels.
3. Add the appropriate volume of cells to each culture vessel and incubate at 37°C,
5% CO2 and 90% humidity.
4. Re-feed the rat fetal NSC cultures every 2−3 days with fresh complete StemPro® NSC
SFM. The morphology of rat fetal NSCs should exhibit short stellate-like processes with
uniform density (see Figure 1 below).
Figure 1 Rat fetal NSCs at passage 3 in adherent culture using StemPro® NSC SFM.
5. When cells reach 75–90% confluency (3–4 days after seeding), the rat fetal NSC cultures
are ready to be passaged.
6. Rinse the culture vessel once with D-PBS without calcium and magnesium, then
remove the medium.
7. Add pre-warmed StemPro® Accutase® and let the cells detach from the culture surface
(within approximately 30 seconds).
8. After detachment, gently pipet the cells up and down to break the clumps into a
uniform cell suspension and add four volumes of complete StemPro® NSC SFM to the
culture vessel.
9. Disperse the cells by pipetting over the culture surface several times to generate a
homogenous cell solution.
10. Transfer the cells to a sterile centrifuge tube and centrifuge at 300 × g for 4 minutes at
room temperature. Aspirate and discard the medium.
11. Resuspend the cell pellet in a minimal volume of pre-warmed complete StemPro® NSC
SFM and remove a sample for counting.
12. Determine the total number of cells and percent viability using trypan blue stain or the
LIVE/DEAD® Cell Vitality Assay Kit.
13. Add enough complete StemPro® NSC SFM to tube for a final cell solution of 1 × 106
viable cells/mL. Incubate at 37°C, 5% CO2 and 90% humidity. Rat fetal NSC cultures
should not be maintained for more than 3 passages.
Important: If you are re-feeding rat fetal NSC in a growth medium other than complete
StemPro® NSC SFM, ensure that the medium is supplemented with 10 ng/mL bFGF to
maintain the undifferentiated state of the rat fetal NSCs.
Neurosphere Suspension
Cultures 1. Resuspend the rat fetal NSCs as follows:
• For freshly prepared rat fetal NSCs, after rinsing with D-PBS, resuspend in warmed
complete StemPro® NSC SFM at a cell density of 1 × 107 viable cells/mL.
• For thawed rat fetal NSCs, after determining the viable cell count, resuspend in
warmed complete StemPro® NSC SFM at a cell density of 1 × 107 viable cells/mL.
2. Plate the rat fetal NSCs onto uncoated or low-attachment culture vessels at a density of
2 × 105 viable cells/cm2. See the table below for recommended seeding densities.
18
Neural Cell Culture and Differentiation
3. Add the appropriate volume of cells to each culture vessel and incubate at 37°C,
5% CO2 and 90% humidity.
4. Carefully re-feed the neurosphere suspension of rat fetal NSCs every 2−3 days with
fresh complete StemPro® NSC SFM without removing any developing neurospheres.
The morphology of the neurospheres should exhibit spherical and transparent multi-
cellular complexes (see Figure 2).
Figure 2 Rat fetal NSCs at passage 3 in neurosphere culture using StemPro® NSC SFM.
5. When the neurospheres reach a diameter of 3.5 mm or larger, the rat fetal NSCs are
ready to be passaged.
6. Transfer the neurosphere suspension into a sterile centrifuge tube and let the
neurospheres settle by gravity or centrifuge at 200 × g for 2 minutes. Aspirate the
supernatant carefully to leave the neurospheres in a minimal volume of medium.
7. Rinse the neurospheres once with D-PBS without calcium and magnesium and leave a
minimal volume of D-PBS.
9. After incubation, gently pipette the cells up and down to get a single-cell suspension
and add 4 mL of complete StemPro® NSC SFM to the tube.
10. Centrifuge at 300 × g for 4 minutes at room temperature, carefully aspirate the
supernatant, resuspend in a minimal volume of pre-warmed complete StemPro® NSC
SFM, and remove a sample for counting on a hemacytometer or Countess® Automated
Cell Counter.
12. Add enough complete StemPro® NSC SFM to the tube for a final cell solution of 1 × 107
viable cells/mL. Incubate at 37°C, 5% CO2 and 90% humidity. Neurosphere suspension
cultures should not be maintained for more than 3 passages.
Important: If you are re-feeding rat fetal NSCs in a growth medium other than
complete StemPro® NSC SFM, ensure that the medium is supplemented with 10 ng/mL
bFGF to maintain the undifferentiated state of the rat fetal NSCs.
Gibco® Neurobiology Protocols Handbook | 19
Xeno-free Culture of Neural Stem Cells
Summary
Neural stem cells (NSCs) derived from human embryonic stem cells (hESCs) have the
potential to help provide understanding for human neurogenesis and for potential
cell therapy applications to treat Parkinson’s Disease or spinal cord injuries. Standard
methods of culturing NSCs raise concerns about pathogen cross-transfer from non-
human sources or contamination with non-neural cells, limiting the efficiency and
specificity of the differentiation protocols. These concerns have led to the development
of xenofree conditions for maintaining and expanding NSCs, which are described in
this protocol.
Required Materials
CELLstart™ CTS™-Coated
Vessels Prepare culture dishes/flasks with CELLstart™ CTS™ as described below.
1. Dilute CELLstart™ CTS™ 1:100 in D-PBS with calcium and magnesium (e.g., 50 μL of
CELLstart™ into 5 mL of D-PBS).
20
Neural Cell Culture and Differentiation
2. Coat the surface of the culture vessel with the working solution of CELLstart™ CTS™
(2.5 mL for a T-25 flask or 60-mm dish, 1.5 mL for a 35-mm dish).
3. Incubate the culture vessel at 37°C in a humidified atmosphere of 5% CO2 for 1 hour.
4. Use the dish immediately after incubation. Aspirate the CELLstart™ CTS™ solution
immediately before use.
Note: Prepare a freshly coated culture vessel each time before plating cells. There is no
need to rinse the culture vessel before use.
Culture Medium Prepare 50 mL of culture medium as follows. The growth factors can be added
immediately before use. After the medium has been supplemented with growth
factors, aliquot the amount needed for the day and store the remaining medium at 4°C.
Formulated medium is stable for 2 weeks if properly stored at 4°C.
Component Amount
Neurobasal® Medium 49 mL
GlutaMAX™-I 2 mM
®
B-27 Supplement XenoFree 1 mL
bFGF 20 ng/mL
EGF 20 ng/mL
Methods
Thawing and Seeding NSCs 1. Remove a vial of cells from liquid nitrogen and quickly thaw the vial in a 37°C water
bath, being careful not to immerse the vial above the level of the cap.
2. When just a small crystal of ice remains, sterilize the outside of the vial with 95%
ethanol. Allow the ethanol to evaporate before opening the vial in a cell culture hood.
3. Gently pipet the cell suspension up and down once, and place it into a 15-mL centrifuge
tube.
4. Add 10 mL of warm culture medium to the tube dropwise to reduce osmotic shock.
7. Seed the cells at a concentration of >90,000 cells/cm2 onto a dish or flask that has been
treated with CELLstart™ CTS™ solution. (Aspirate the CELLstart™ CTS™ solution
immediately before using the dish or flask.)
Culture and Propagation 1. Twenty-four hours after seeding the cells, replace the culture medium.
2. Replace the spent medium every other day with an equal volume of fresh culture
medium.
Note: If the medium turns yellow, change the medium daily. Yellow medium will affect
the NSC proliferation rate.
4. To split the cell culture 1:2, aspirate the medium and wash the cells twice with 5 mL of
D-PBS (without calcium and magnesium).
5. Add 1 mL of TrypLE™ Select to dissociate the cells, and incubate for 2 minutes at 37°C.
6. Add 4 mL of culture media to neutralize the TrypLE™ Select activity and pipet up and
down 2–3 times to get a uniform cell suspension. Check the cells under a microscope.
10. Split the cell suspension into two fresh T-25 flasks that have been treated with
CELLstart™ CTS™ solution. Seed each flask with 5 mL of cell suspension.
11. Incubate the flasks at 37°C in a humidified atmosphere (90%) of 5% CO2 in air.
12. Grow the cells until semi-confluent, changing the medium once after 12 hours and
every two days thereafter.
13. Passage the cells when the culture reaches ~80% confluence.
Figure 1 Phase contrast microphotograph showing NSCs cultured in xenofree media 24 hours post-thaw
(Panel A) and semi-confluent NSCs cultured in xenofree media for 3 days (Panel B).
A B
22
Neural Cell Culture and Differentiation
Summary
The protocols in this chapter describe the steps involved in differentiating neural stem
cells (NSC) to neurons, astrocytes, and oligodendrocyte lineages in vitro. NSCs are self-
renewing multipotent stem cells that can be proliferated in vitro in supportive culture
systems such as Stempro® NSC SFM and can further be differentiated into downstream
lineages. The protocols described are primarily optimized with NSCs derived from
human embryonic stem cells (ESC) or induced pluripotent stem cells (iPSC). Some
optimization in terms of reagent concentration and duration of in vitro differentiation
is expected for NSCs from other species such as rat or mouse, as well as with NSCs
derived from patient-specific iPSCs.
Required Materials
Cells • GIBCO® Human Neural Stem Cells (H9 hESC-Derived) (Cat. no. N7800)
Secondary Antibodies Ex/Em* (color) Alexa® no. 2nd host 2nd against Cat. no. Concentration
Goat Mouse IgM A31552 1:1,000
Goat Mouse IgG A21049 1:1,000
346/442 (Blue) 350 Goat Rat IgG A21093 1:1,000
Goat Rabbit IgG A21068 1:1,000
Donkey Goat IgG A21081 1:1,000
Goat Mouse IgM A21042 1:1,000
Goat Mouse IgG A11029 1:1,000
Goat Rat IgM A21212 1:1,000
495/519 (Green) 488
Goat Rat IgG A11006 1:1,000
Goat Goat IgG A11034 1:1,000
Donkey Mouse IgM A11055 1:1,000
Goat Mouse IgM A21044 1:1,000
Goat Mouse IgG A11029 1:1,000
Goat Rat IgM A21213 1:1,000
590/617 (Red) 594
Goat Rat IgG A11007 1:1,000
Goat Rabbit IgG A11037 1:1,000
Donkey Goat IgG A11058 1:1,000
Goat Mouse IgM M31504 1:500
496, 536, 565/576
NA Goat Mouse IgG P852 1:1,000
(Red)
Goat Rabbit IgG P2771MP 1:1,000
*Approximate excitation and emission maxima, in nm; NA = not applicable.
24
Neural Cell Culture and Differentiation
Preparing Media
1. Reconstitute bFGF and EGF with 0.1% BSA solution (in KnockOut™ D-MEM/F-12) at
a concentration of 100 μg/mL. You will need 20 μL of each per 100 mL of complete
medium. Freeze unused portions in aliquots.
2. Mix the following components under aseptic conditions. For larger volumes, increase
the component amounts proportionally: If desired, add 1 mL of Antibiotic-Antimycotic
solution per 100 mL of complete medium.
KnockOut™ D-MEM/F-12 1X 97 mL
GlutaMAX™-I Supplement 2 mM 1 mL
You may observe a white precipitate when thawing StemPro® Neural Supplement; this
precipitate will disappear when the supplement is completely thawed or dissolved.
Neural Differentiation
Medium Neural differentiation medium requires supplementation of Neurobasal® medium with
B-27® Serum-Free Supplement and GlutaMAX™-I. Neural differentiation medium is
stable for 2 weeks when stored in the dark at 2-8°C.
Neurobasal® Medium 1X 97 mL
GlutaMAX™-I Supplement 2 mM 1 mL
If faster differentiation is desired, add dibutyryl cAMP (Sigma, Cat. no. 0627) to a final
concentration of 0.5 mM at day 7 for a duration of 3 days, as indicated in the differentitation
protocols.
Astrocyte Differentiation
Medium Astrocyte differentiation medium requires supplementation of D-MEM with N-2,
GlutaMAX™-I, and FBS. Astrocyte differentiation medium is stable for 4 weeks when
stored in the dark at 2-8°C.
D-MEM 1X 97 mL
N-2 Supplement 1% 1 mL
GlutaMAX™-I Supplement 2 mM 1 mL
FBS 1% 1 mL
Oligodendrocyte
Differentiation Medium Oligodendrocyte differentiation medium requires supplementation of Neurobasal®
medium with B-27®, GlutaMAX™-I, and T3. Oligodendrocyte differentiation medium is
stable for 2 weeks when stored in the dark at 2-8°C.
Neurobasal® Medium 1X 97 mL
GlutaMAX™-I Supplement 2 mM 1 mL
* You can prepare a 30 μg/mL T3 stock solution (1,000X) in distilled water. Filter
sterilize the T3 stock solution.
26
Neural Cell Culture and Differentiation
Preparing Matrix
2. Coat the surface of the culture vessel with the working solution of CELLStart™ CTS®
(14 mL for T-75, 7 mL for T-25, 3.5 mL for 60-mm dish, 2 mL for 35-mm dish).
3. Incubate the culture vessel at 37°C in a humidified atmosphere of 5% CO2 in air for
1 hour.
4. Remove the vessel from the incubator and store it until use. Immediately before use,
remove all CELLStart™ CTS® solution and replace it with complete StemPro® NSC SFM.
Note: You may coat the plates in advance and store them at 4°C, wrapped tightly with
Parafilm®, for up to 2 weeks. Do not remove CELLStart™ CTS® solution until just prior
to use. Make sure the plates do not dry out.
2. Thaw 1 tube of Geltrex™ (0.5 mL, aliquoted as above) slowly at 4°C, and add 49.5 mL of
cold D-MEM/F-12 (1:100 dilution). Mix gently.
3. Cover the whole surface of each culture plate with the Geltrex™ solution (1.5 mL for a
35-mm dish, 3 mL for 60-mm dish, 5 mL for a T-25 culture flask).
4. Seal each dish with Parafilm® to prevent drying, and incubate 1 hour at room
temperature in a laminar flow hood.
5. Immediately before use, remove all Geltrex™ solution, wash once with D-PBS with
calcium and magnesium, and replace pre-warmed complete medium.
Note: You may store the Geltrex™-treated dish at 4°C, wrapped tightly with Parafilm®,
for up to 1 month. Do not remove Geltrex™ solution until just prior to use.
2. Thaw the laminin slowly at 2–8°C and prepare 10 μg/mL working solution in cell
culture-grade distilled water. Aliquot the working solution into polypropylene tubes,
and store the tubes at –20°C until use. Avoid repeated freeze/thaw cycles.
3. Dilute the poly-L-ornithine stock solution 1:500 in cell culture-grade distilled water to
make 20 μg/mL working solution.
4. Coat the surface of the culture vessel (with or without cover slips) with the
poly‑L‑ornithine working solution (14 mL for T-75, 7 mL for T-25, 3.5 mL for 60-mm
dish, 2 mL for 35-mm dish).
7. Coat the surface of the culture vessel (with or without cover slips) with the laminin
working solution (14 mL for T-75, 7 mL for T-25, 3.5 mL for 60-mm dish, 2 mL for
35‑mm dish).
9. Rinse the culture vessel with D-PBS without calcium or magnesium, and store the
vessel covered with D-PBS until use. Immediately before use, remove all D-PBS and
replace it with complete StemPro® NSC SFM.
Note: You may coat the plates in advance and store them at room temperature,
wrapped tightly with Parafilm®, for up to 1 week. Do not remove D-PBS until just prior
to use. Make sure the plates do not dry out.
Differentiation into
Neurons 1. Plate neural stem cells on a polyornithine and laminin-coated culture dish in complete
StemPro® NSC SFM at 2.5 × 104–5 × 104 cells/cm2.
2. After 2 days, change the medium to neural differentiation medium. Change the spent
medium every 3–4 days.
3. If expedited differentiation is desired, add 0.5 mM of dibutyryl cAMP (Sigma, Cat. no.
D0627) to the differentiation medium daily starting at day 7 of differentiation for 3 days.
IMPORTANT! Do not expose cells to air at any time after they have differentiated into
neurons.
Differentiation into
Astrocytes 1. Plate the NSCs on a Geltrex™-coated culture dish in complete StemPro® NSC SFM at
2.5 × 104 cells/cm2.
2. After 2 days, change medium to astrocyte differentiation medium. Change the spent
medium every 3–4 days.
28
Neural Cell Culture and Differentiation
Differentiation into
Oligodendrocytes 1. Plate the NSCs on a polyornithine and laminin-coated culture dish in complete
StemPro® NSC SFM at 2.5 × 104–5 × 104 cells/cm2.
Preparing
Paraformaldehyde Fixing
Solution 20% paraformaldehyde (PFA) stock solution
2. Add 0.25 mL of 10 N NaOH and heat the solution at 60°C using a magnetic stirrer until
the solution is completely dissolved.
3. Filter the solution through a 0.22-μm filter, and cool on ice. Make sure the pH is 7.5–8.0.
4. Aliquot 2 mL in 15-mL tubes, freeze the tubes on dry ice, and store them at –20°C.
1. Add 8 mL of PBS into each 15-mL tube containing 2 mL of 20% PFA, and thaw each
tube in a 37°C water bath.
Fixing Cells 1. Remove culture medium and gently rinse the cells once with D-PBS, without dislodging
the cells.
2. Fix the cells with 4% fresh Paraformaldehyde Fixing Solution (PFA) at room
temperature for 15 minutes.
5. Proceed to staining, described below. You may store slides for up to 3–4 weeks in
D-PBS at 4°C before staining. Do not allow slides to dry.
Staining Cells 1. Incubate cells for 30–60 minutes in blocking buffer (5% serum of the secondary antibody
host species, 1% BSA, 0.1% Triton®-X in D-PBS with Ca2+ and Mg2+).
Note: If you are using a surface antigen such as GalC, omit Triton®-X from the blocking
buffer.
2. Remove the blocking buffer and incubate the cells overnight at 4°C with primary
antibody diluted in 5% serum. Ensure that the cell surfaces are covered uniformly with
the antibody solution.
3. Wash the cells 3X for 5 minutes with D-PBS containing Ca2+ and Mg2+ (if using a slide,
use a staining dish with a magnetic stirrer).
4. Incubate the cells with fluorescence-labeled secondary antibody (5% serum in D-PBS
with Ca2+ and Mg2+) in the dark at 37°C for 30–45 minutes.
5. Wash the cells 3X with D-PBS containing Ca2+ and Mg2+, and in the last wash, counter
stain the cells with DAPI solution (3 ng/mL) for 5–10 minutes, and rinse with D-PBS.
6. If desired, mount using 3 drops of ProLong® Gold antifade reagent per slide and seal
with the cover slip. You may store the slides in the dark at 4°C.
Expected Results
Figure 1 Fluorescence images (20X) of GIBCO® hNSCs that have been cultured in StemPro® NSC SFM
for three passages, and then allowed to differentiate into neurons, oligodendrocytes, or astrocytes. Upon
directed differentiation, cells start to lose the undifferentiated NSC marker, nestin, but stain positive for
the differentiated cell type markers Dcx, GalC, and GFAP. Cells were stained for the undifferentiated NSC
markers nestin (red) and SOX2 (green) prior to directed differentiation (Panel A). Cell were then differentiated
into neurons and glial cells, and respectively stained for the neuronal marker Dcx (green) (Panel B), for
the oligodendrocyte marker GalC (red) (Panel C), or for the astrocyte marker, GFAP (green) (Panel D). The
nuclei were counterstained with DAPI (blue) in panels B–D.
A B
C D
30
Neural Cell Culture and Differentiation
Troubleshooting
The table below lists some causes and solutions to help you troubleshoot your potential
differentiation problems.
Cells have been passaged too many times Obtain new GIBCO® human neural stem cells
References
Trujillo, C.A., Schwindt, T.T., Martins, A.H., Alves, J.M., Mello, L.E., and Ulrich, H.
2009. Novel perspectives of neural stem cell differentiation: from neurotransmitters to
therapeutics. Cytometry A 75:38–53.
Elkabetz, Y. and Studer, L. 2008. Human ESC-derived neural rosettes and neural stem
cell progression. Cold Spring Harb Symp Quant Biol. 73:377–387.
Summary
Glial precursor cells (GPCs), also known as glial restricted progenitors (GRP) or
oligodendrocyte progenitor cells (OPCs), are cells that have the potential to differentiate
into oligodendrocytes or astrocytes. The GPC population is derived from tissue or is
generated from pluripotent cells by differentiation, which is induced by exogenously
applied factors. Here we described a culture system that can be adjusted to favor
differentiation into either astrocytes or oligodendrocytes.
Required Materials
32
Neural Cell Culture and Differentiation
Preparing Media
Preparing Oligodendrocyte
Differentiation Medium Oligodendrocyte differentiation medium uses Neurobasal® medium supplemented
with B-27®, GlutaMAX™-I, and T3. Complete medium is stable for 2 weeks when stored
in the dark at 2-8°C.
To prepare 100 mL of complete medium, mix the following components under aseptic
conditions. For larger volumes, increase the component amounts proportionally.
GlutaMAX™-I Supplement 2 mM 1 mL
®
B-27 Supplement 2% 2 mL
T3 30 ng/mL 0.1 mL
Preparing Astrocyte
Differentiation Medium Astrocyte differentiation medium uses D-MEM medium supplemented with N-2,
GlutaMAX™-I, and FBS. Complete medium is stable for 2 weeks when stored in the
dark at 2-8°C.
D-MEM Medium 1X 97 mL
GlutaMAX™-I Supplement 2 mM 1 mL
N-2 Supplement 1% 1 mL
FBS 1% 1 mL
Preparing Matrix
3. Incubate the culture vessel at 37°C in a humidified atmosphere of 5% CO2 for at least
1 hour.
6. Add 2 mL of 1 μg/mL laminin solution to a 35-mm dish (0.5 mL for 4-well plate or
slide, 0.25 mL for 8-well slide).
7. Incubate the culture vessel at 37°C in a humidified atmosphere of 5% CO2 for at least
1 hour. Store it at 4°C until use.
Note: You may coat the plates in advance and store them at 2-8°C, wrapped tightly
with Parafilm®, for up to 4 weeks.
2. Thaw one tube of Geltrex™ (1 mL aliquot described above) slowly at 4°C, and add
49 mL of cold D-MEM/F-12. Mix gently.
3. Cover the whole surface of each culture plate with the Geltrex™ solution (1.5 mL for a
35-mm dish, 3 mL for 60-mm dish, 5 mL for a T-25 culture flask).
4. Seal each dish with Parafilm® to prevent drying, and incubate 1 hour at room
temperature in a laminar flow hood. Store at 4°C until use.
5. Immediately before use, remove all Geltrex™ solution, wash once with D-PBS with
calcium and magnesium, and replace it with pre-warmed complete medium.
Note: You may store the Geltrex™-treated dish at 2-8°C, wrapped tightly with
Parafilm®, for up to 1 month. Ensure that plates do not dry out. Do not remove the
Geltrex™ solution until just prior to use.
Differentiation of GRPs
Differentiation to
Oligodendrocytes 1. Plate glial precursor cells on poly-L-ornithine and laminin coated plate in StemPro®
NSC SFM at a density of 2.5 × 104-5 × 104 cells/cm2.
2. Culture the cells for 2 days, then change the medium to Oligodendrocyte Differentiation
Medium.
Differentiation to
Astrocytes 1. Plate glial precursor cells on Geltrex™ coated plate in StemPro® NSC SFM at a density of
2.5 × 104 cells/cm2.
2. Culture the cells for 2 days, then change th emedium to Astrocyte Differentiation
Medium.
Summary
Astrocytes are the most numerous cell type in the central nervous system (CNS). They
play critical roles in adult CNS homeostasis, provide biochemical and nutritional
support of neurons and endothelial cells that form the blood-brain barrier, perform
the vast majority of synaptic glutamate uptake, and maintain extracellular potassium
levels. Astroglial dysfunction has been implicated in a number of CNS pathologies. This
protocol describes the preparation of primary cortical astrocytes from new-born rats or
mice.
Required Materials
Astrocyte Medium GIBCO® Astrocyte Medium has been specifically formulated for the growth and
maintenance of human and rat astrocytes while retaining their phenotype. The medium
has three components: basal medium (D-MEM), N-2 Supplement, and One Shot™ Fetal
Bovine Serum (FBS). Epidermal growth factor (EGF) may also be added to enhance
astrocyte proliferation.
To prepare 100 mL of complete medium, mix the following components under aseptic
conditions. For larger volumes, increase the component amounts proportionally.
Component Amount
D-MEM 83 mL
N-2 15 mL
FBS 1 mL
Penicillin-streptomycin 1 mL
Collagen Prepare a 50 μg/mL working solution in distilled water with 0.02 M acetic acid and
sterilize the solution with a 0.22-μm filter.
Dibutyryl cyclic-AMP
(dBcAMP) Prepare a 0.25 M stock solution of dBcAMP in D-PBS, aliquot into sterilized tubes, and
store at –20°C.
1. Coat the culture vessels with collagen and let stand for 45 minutes at room temperature.
Rinse with D-PBS without calcium or magnesium two times.
2. Anesthetize rat or mouse pups with ether in a desiccator in a chemical fume hood.
3. Remove pups from the hood and spray 70% ethanol over the animal. Decapitate the
animals with scissors. Open the skull with iridectomy scissors. Remove the meninges
and dissect the brain tissue from the cortices.
4. Put the cortices in a petri dish containing 5−10 mL of ice-cold HBSS. Pool the cortices
from two pups in a new petri dish and wash with 5 mL of HBSS.
36
Neural Cell Culture and Differentiation
5. Take the petri dish to a laminar flow hood. Mince the cortices into small pieces with a
scissors in a petri dish containing about 5 mL of ice-cold HBSS. Transfer the tissue to a
15-mL sterile tube. Centrifuge the tube at 200 × g for 3 minutes at 4°C and aspirate the
supernatant.
6. Resuspend the tissue in 5 mL of 0.05% trypsin and incubate at 37°C for 25 minutes in a
shaker bath.
7. Centrifuge the tissue suspension at 200 × g for 3 minutes, aspirate the trypsin solution
with a pipette, and rinse the cells 3 times with 3 mL of HBSS.
8. Add 6 mL of astrocyte medium and pipet the cell suspension up and down with a
10‑mL pipette to dissociate cells.
9. Filter the cell suspension through a 70-μm mesh cell strainer into a 50-mL sterile tube.
Rinse the mesh with another 4 mL of astrocyte medium (total of 10 mL suspension).
11. Determine the total number of cells and percent viability using trypan blue stain or the
LIVE/DEAD® Cell Vitality Assay Kit.
12. Dilute the cell suspension to 5 × 104 viable cells/mL with astrocyte medium and plate
the cells into culture vessels at 2.5 × 104/cm2.
13. Incubate the cells in a 37°C incubator with 5% CO2 and 90% humidity.
14. Change the astrocyte medium the next day and then every other day until cells are
confluent.
15. When confluent, feed the cells with astrocyte medium containing 0.25 mM dBcAMP to
induce differentiation. (Dilute 0.25 M stock of dBcAMP 1:1,000 in astrocyte medium.)
16. Feed the cultures with dBcAMP two times per week and check for differentiation.
17. Astrocytes are ready for experiments 2−3 weeks after culturing.
Summary
The ability to culture primary neurons under serum-free conditions facilitates tighter
control of neuronal studies. Some serum-free media and supplements allow for the
low-density neuronal cultures, which in turn enables the study of individual neurons
and synapses. This has not been possible using serum–supplemented media without
a feeder layer of glial cells. In serum-supplemented media, glial cells continue to
multiply, necessitating the use of cytotoxic mitotic inhibitors. Serum also contains
unknown and variable levels of growth factors, hormones, vitamins, and proteins.
This chapter details the isolation and culture of neural cells in serum-free media and
supplements.
Required Materials
Culturing Embryonic
Neurons • Poly-D-lysine hydrobromide (Sigma, Cat. no. P-6407)
• 48-well plate or 8-chambered slides
• Distilled water (Cat. no. 15230-162)
• Dulbecco’s Phosphate-Buffered Saline (D-PBS) (Cat. no. 14040-141)
• Neurobasal® Medium (Cat. no. 21103-049)
• B-27® Serum-Free Supplement (Cat. no. 17504)
• GlutaMAX™-I (Cat. no. 35050)
38
Neural Cell Culture and Differentiation
Preparing Media
Hibernate®-E Complete
Medium Hibernate®-E is a serum-free, nutrient basal medium for the short-term maintenance of
cultured rat neurons and long-term storage of viable brain tissue in ambient CO2 (0.2%)
conditions. The complete medium consists of Hibernate®-E medium supplemented with
B-27® Serum-Free Supplement and GlutaMAX™-I. Hibernate®-E complete medium is
stable for 2 weeks when stored in the dark at 2–8°C.
Neurobasal® Complete
Medium Neurobasal® complete medium requires supplementation of Neurobasal® medium
with B-27® Serum-Free Supplement and GlutaMAX™-I. Complete medium is stable for
2 weeks when stored in the dark at 2–8°C.
Neurobasal® Medium 1X 98 mL
For primary rat hippocampus neuron cultures, Neurobasal® complete medium requires
additional supplementation with 25 µM L-Glutamate up to the fourth day of culture.
Preparing Matrix
2. Dilute the poly-D-lysine stock solution 1:40 in D-PBS to prepare a 50 μg/mL working
solution (i.e., 125 μL of poly-D-lysine stock solution into 5 mL of D-PBS).
3. Coat the surface of the culture vessel with the working solution of poly-D-lysine
(150 μL/cm2, i.e., 100 μL per well for a 48-well plate).
5. Remove the poly-D-lysine solution and rinse 3 times with distilled water. Make sure
to rinse the culture vessel thoroughly, because excess poly-D-lysine can be toxic to the
cells.
6. Leave the coated vessels uncovered in the laminar hood until the wells have completely
dried. You may use the dry plates immediately or store them at 4°C, wrapped tightly
with Parafilm®, for up to one week.
Isolating Neurons
1. Dissect cortex or hippocampus pairs from ten E-18 rat embryo brains. Remove all the
meninges thoroughly.
2. Collect all the tissue in a conical tube containing Hibernate®-E supplemented with
2% B-27® Serum-Free Supplement and 0.5 mM GlutaMAX™-I at 4°C.
3. Allow the tissue to settle to the bottom of the tube and then carefully remove the
supernatant leaving only the tissue covered by the medium.
5. Add 6 mL of complete Hibernate®-E medium to the tube and centrifuge for 5 minutes at
150 × g.
7. Let the tube stand undisturbed for 2 minutes to allow for the cell debris (if any) to settle
down.
8. Transfer the cells to a new tube leaving behind all the debris.
9. Count the cells using a hemacytometer, cell counter and trypan blue, or the Countess®
Automated Cell Counter.
40
Neural Cell Culture and Differentiation
11. Remove the supernatant and resuspend the cell pellet in Neurobasal® medium with
2% B-27® Serum-Free Supplement and 0.5 mM GlutaMAX™-I for culturing.
Note: Plate the cells immediately after resuspension. If you need to store the cells
longer, store them in Hibernate®-E medium supplemented with 2% B-27® Serum-Free
Supplement and 0.5 mM GlutaMAX™-I at 4°C for up to 48 hours. Do not expose the
neurons to air at any time.
Culturing Neurons
1. Plate ~1 × 105 cells per well in poly-D-lysine coated 48-well plate or an 8-chambered
slide. Bring the cell suspension volume to 500 μL per well by adding complete
Neurobasal® medium.
3. Feed the cells every third day by aspirating half of the medium from each well and
replacing it with fresh medium.
Preparing
Paraformaldehyde Fixing
Solution 20% paraformaldehyde (PFA) stock solution
1. Add PBS to 20 g of EM grade paraformaldehyde, and bring the volume up to 100 mL.
2. Add 0.25 mL of 10 N NaOH and heat the solution at 60°C using a magnetic stirrer until
the solution is completely dissolved.
3. Filter the solution through a 0.22-μm filter, and cool on ice. Make sure the pH is 7.5–8.0.
4. Aliquot 2 mL in 15-mL tubes, freeze the tubes on dry ice, and store them at –20°C.
1. Add 8 mL of PBS into each 15-mL tube containing 2 mL of 20% PFA, and thaw each
tube in a 37°C water bath.
Fixing Cells 1. Remove the culture medium and gently rinse the cells without dislodging them twice
with D-PBS containing Ca2+ and Mg2+.
2. Fix the cells with 4% fresh Paraformaldehyde Fixing Solution (PFA) at room
temperature for 15 minutes.
3. Rinse the cells three times with D-PBS containing Ca2+ and Mg2+.
4. Permeabilize the cells with 0.3% Triton®-X (diluted in D-PBS with Ca2+ and Mg2+) for
5 minutes at room temperature.
5. Rinse the cells three times with D-PBS containing Ca2+ and Mg2+.
Staining Cells 1. Incubate cells in 5% goat serum diluted in D-PBS with Ca2+ and Mg2+ for 60 minutes at
room temperature.
2. Remove the 5% goat serum solution and incubate the cells overnight with the primary
antibody (Mouse anti-MAP2 at 10 μg/mL and/or Rabbit anti-GFAP at 4 μg/mL)
diluted in 5% goat serum at 4°C. Ensure that the cell surfaces are covered uniformly
with the antibody solution.
3. Wash the cells three times for 5 minutes with D-PBS containing Ca2+ and Mg2+ (if using
a slide, use a staining dish with a magnetic stirrer).
4. Incubate the cells with fluorescence-labeled secondary antibody (Alexa Fluor® 488
goat‑anti mouse (H+L) at 10 μg/mL and/or Alexa Fluor® 594 goat-anti rabbit (H+L) at
10 μg/mL) diluted in 5% goat serum solution for 60 minutes at room temperature.
5. Wash the cells three times with D-PBS containing Ca2+ and Mg2+. In the last wash,
counter-stain the cells with DAPI solution (3 ng/mL) for 10 minutes.
6. Rinse the cells with D-PBS, and if desired, mount using 3 drops of ProLong® Gold
antifade reagent per slide and seal it with the cover slip. You may store the slides in the
dark at 4°C.
7. Observe the cells under the microscope using filters for FITC, Cy5, and DAPI.
42
Neural Cell Culture and Differentiation
Expected Results
Figure 1 Primary Rat Cortical neurons. Immunofluorescence detection of primary neuronal cells stained
with mouse anti-MAP2 antibody (Green) and presence of astrocytes as detected by rabbit anti-GFAP marker
(Red). Nuclei are stained with DAPI (blue).
Troubleshooting
The procedures are designed for neuronal cells grown in Neurobasal® medium
supplemented with 2% B-27® Serum-Free Supplement and 0.5 mM GlutaMAX™-I .
Results may differ with culture systems grown in other complete media formulations,
which can result in higher numbers of cells other than neurons (i.e., astrocytes).
References
Brewer, G. J. and Price, P.J. 1996. Viable cultured neurons in ambient carbon dioxide
and hibernation storage for a month. Neuroreport 7:1509–1512.
Brewer, G. J., Torricelli, J.R., Evege, E. K., and Price, P.J. 1993. Optimized survival of
hippocampal neurons in B27-supplemented Neurobasal, a new serum-free medium
combination. J Neurosci Res. 35:567–576.
Summary
Directed differentiation of specific lineages has been a focal point in the field of human
embryonic stem cell (hESC) research. Cell replacement therapy using hESCs have the
potential for treating Parkinson’s disease and other neurodegenerative disorders. This
chapter describes the procedure for the derivation of dopaminergic (DA) neurons from
hESCs.
Required Materials
Cells • GIBCO® Mouse Embryonic Fibroblasts (MEF), irradiated (Cat. no. S1520-100)
• Human embryonic stem cells (hESC)
Media and Reagents • Dulbecco’s Phosphate-Buffered Saline (D-PBS) without Ca2+ and Mg2+ (Cat. no. 14190)
• D-MEM/F-12 with GlutaMAX™-I (Cat. no. 10565-018)
• Neurobasal® Medium (Cat. no. 21103-049)
• Knockout™ Serum Replacement (Cat. no. 10828-028)
• 10% Bovine Serum Albumin (BSA) (Cat. no. P2489)
• Fetal Bovine Serum (FBS) (Cat. no. 16000-044)
• Dulbecco’s Modified Eagle Medium (D-MEM) (Cat. no. 10569-010)
• Non-essential Amino Acids Solution (NEAA) (Cat. no. 11140)
• B-27® Supplement without Vitamin A (Cat. no. 12587-010)
• N-2 supplement (Cat. no. 17502-048)
• β-Mercaptoethanol (Cat. no. 21985-023)
• Attachment Factor (Cat. no. S-006-100)
• Natural Mouse Laminin (Cat no. 23017-015)
• StemPro® Accutase® Cell Dissociation Reagent (Cat. no. A11105-01)
• Recombinant Human FGF Basic (bFGF) (Cat. no. 13256-029)
• FGF-8b Recombinant Human (Cat. no. PHG0271)
• B-DNF Recombinant Human (Cat. no. PHC7074)
• G-DNF Recombinant Human (Cat. no. PHC7045)
• Trypan Blue Stain (Cat. no. 15250-061)
• Distilled water (Cat. no. 15230-162)
• Poly-L-Ornithine (Sigma, Cat. no. P3655)
• Heparin (Sigma, Cat. no. H3149)
• Ascorbic Acid (Sigma, Cat. no. A4403)
• Dibutyryl cyclic-AMP (dcAMP) (Sigma, Cat. no. D0627)
• Recombinant human sonic hedgehog (SHH) (R&D systems, Cat. no. 1314-SH-025)
44
Neural Cell Culture and Differentiation
Special Tools • StemPro® EZPassage™ Disposable Stem Cell Passaging Tool (Cat. no. 23181-010)
• Cell scraper (Fisher, Cat. no. 087711A)
Preparing Media
Heparin
Prepare a 2-mg/mL stock solution in D-PBS, aliquot 0.5 mL into sterilized tubes, and
store at –80°C.
Ascorbic acid
Prepare a 200-mM stock solution in D-PBS, aliquot 0.5 mL into sterilized tubes, and
store at –20°C.
Poly-L-Ornithine
Prepare a 10-mg/mL stock solution in distilled water, aliquot 0.5 mL into sterilized
tubes, and store at –20°C.
Mouse Embryonic
Fibroblast (MEF) Medium To prepare 100 mL of MEF medium, aseptically mix the following components. For
larger volumes, increase the component amounts proportionally.
Component Amount
D-MEM 90 mL
FBS 10 mL
Component Amount
D-MEM/F-12 79 mL
NEAA 1 mL
β-Mercaptoethanol* 182 μL
Neural Induction Medium To prepare 100 mL of neural induction medium, aseptically mix the following
components. For larger volumes, increase the component amounts proportionally.
Neural induction medium lasts for up to 7 days at 4°C.
Component Amount
D-MEM/F-12 98 mL
N-2 Supplement 1 mL
NEAA 1 mL
46
Neural Cell Culture and Differentiation
Neural Expansion Medium To prepare 100 mL of neural expansion medium, aseptically mix the following
components. For larger volumes, increase the component amounts proportionally.
Neural expansion medium lasts for up to 7 days at 4°C.
Component Amount
D-MEM/F-12 96 mL
N-2 Supplement 1 mL
B-27® Supplement 2 mL
NEAA 1 mL
DA Neuronal Differentiation
Medium To prepare 100 mL of DA neural differentiation medium, aseptically mix the following
components. For larger volumes, increase the component amounts proportionally. DA
neural differentiation medium lasts for up to 7 days at 4°C.
Component Amount
Neurobasal® Medium 96 mL
L-Glutamine 1 mL
B-27® Supplement 2 mL
NEAA 1 mL
Thawing MEFs 1. Wearing eye protection and ultra low-temperature cryo-gloves, remove the vials of
irradiated MEF from the liquid nitrogen storage tank using metal forceps.
Note: Transfer the vials into a container with a small amount of liquid nitrogen if the
vials are exposed to ambient temperature for more than 15 seconds between removal
and step 3.
2. Briefly roll the vials containing MEF between your hands for about 10–15 seconds to
remove frost and swirl them gently in a 37°C water bath. Do not submerge the vials
completely.
3. When only a small amount of ice remains in the vials, remove them from the water
bath. Spray the outside of the vials with 70% ethanol before placing them in the cell
culture hood.
4. Pipet the thawed cells gently into a 15-mL conical tube using a 1-mL pipette.
5. Rinse the cryovial with 1 mL of pre-warmed MEF medium. Transfer the medium to the
same 15-mL tube containing the cells.
6. Add 4 mL of pre-warmed MEF medium dropwise to the cells. Gently mix by pipetting
up and down.
Note: Adding the medium slowly helps cells to avoid osmotic shock.
8. Aspirate the supernatant and resuspend the cell pellet in 5 mL of pre-warmed MEF
medium.
9. Remove 10 μL of cell suspension and determine the viable cell count using your method
of choice.
Note: We recommend using the Countess® Automated Cell Counter for easy and
accurate cell counting and viability measurements.
Plating MEFs 1. Centrifuge the MEFs at 200 × g for 5 minutes and aspirate the supernatant.
2. Resuspend the cell pellet in MEF medium to a concentration of 2.5 × 106 cells/mL.
3. Aspirate the Attachment Factor solution from the coated culture vessels and wash the
plates once with D-PBS.
4. Add the appropriate amount of MEF medium into each culture vessel (2.5 mL into each
well of 6-well plate, 5 mL into each 60-mm dish, or 10 mL into each 100-mm dish).
5. Into each of these culture vessels, add the appropriate amount of MEF suspension
(0.1 mL into each well of 6-well plate, 0.2 mL into each 60-mm dish, or 0.6 mL into
each 100-mm dish). The recommended plating density for GIBCO® Mouse Embryonic
Fibroblasts (Irradiated) is 2.5 × 104 cells/cm2.
6. Move the culture vessels in several quick back-and-forth and side-to-side motions to
disperse cells across the surface of the wells and dishes. After plating the cells, place
the vessels in a 37°C incubator with a humidified atmosphere of 5% CO2. Use the MEF
plates and dishes within 3–4 days of plating.
48
Neural Cell Culture and Differentiation
1. Wearing eye protection and ultra low-temperature cryo-gloves, remove a vial of hESCs
from the liquid nitrogen storage tank using metal forceps.
2. Immerse the vial in a 37°C water bath without submerging the cap. Swirl the vial
gently.
3. When only a small amount of ice remains in the vial, remove it from the water bath.
Spray the outside of the vial with 70% ethanol before placing it in the cell culture hood.
4. Transfer the cells gently into a sterile 15-mL conical tube using a 1-mL pipette. Rinse the
vial with 1 mL of pre-warmed hESC medium to collect the remaining cells in the vial
and add them dropwise to the cells in the 15-mL conical tube.
Note: Adding the medium slowly helps cells to avoid osmotic shock.
5. Add 4 mL of pre-warmed hESC medium dropwise to the cells in the 15-mL conical
tube. While adding the medium, gently move the tube back and forth to mix hESCs.
7. Aspirate the supernatant and resuspend the cell pellet in 5 mL of pre-warmed hESC
medium.
8. Label the culture vessel containing inactivated MEFs with the passage number of the
hESCs from the vial, the date and your initials.
9. Aspirate the MEF medium from the culture vessel containing the MEFs and gently add
the resuspended hESCs into the vessel.
10. Move the culture vessel in several quick back-and-forth and side-to-side motions to
disperse the cells across the surface of the vessel. Place the vessel gently into a 37°C
incubator with a humidified atmosphere of 5% CO2.
11. Replace the spent medium and examine the cells under a microscope daily. If feeding
cells in more than one vessel, use a different pipette for each vessel to reduce the risk of
contamination.
Note: hESC colonies may not be visible in the first several days.
12. Observe the hESCs every day and passage the cells whenever the colonies are too big or
too crowded. The ratio of splitting depends on the total number of hESC in the culture
vessel (approximately 1:2 to 1:4 for hESCs at the first time of recovery).
Passaging hESCs
General Guidelines • In general, split cells when the first of the following events occurs:
–– MEF feeder layer is two weeks old.
–– hESC colonies are becoming too dense or too large.
–– Increased differentiation occurs.
• The split ratio varies, but it is generally between 1:4 and 1:6.
• Occasionally hESCs grow at a different rate, requiring the split ratio to be adjusted.
A general rule is to observe the last split ratio and adjust the ratio according to the
appearance of the hESC colonies.
• If the cells look healthy and colonies have enough space, split them using the same
ratio as the previous passage. If the cells are overly dense and crowded, increase the
split ratio; if the cells are sparse, decrease the ratio.
• Generally, hESCs need to be split every 4–10 days based upon their appearance.
Passaging hESCs 1. Two days prior to passaging your hESC culture, prepare fresh MEF culture vessels.
2. Remove the culture vessel containing hESCs from the incubator. Mark differentiated
colonies under a microscope using a microscopy marker and remove them by
aspirating with a Pasteur pipette in the culture hood.
3. Add an appropriate amount of pre-warmed hESC medium into each culture vessel
(2 mL for each 60-mm dish or 4 mL for each 100-mm dish).
4. Roll the StemPro® EZPassage™ Disposable Stem Cell Passaging Tool across the entire
vessel in one direction (left to right). Rotate the culture vessel 90 degrees and roll the
tool across the entire dish again.
5. Using a cell scraper, gently detach the cells off the surface of the culture vessel. Gently
transfer the cell clumps into a 15- or 50-mL conical tube using a 5-mL pipette.
6. Rinse the culture vessel with an appropriate amount of pre-warmed hESC medium
(1 mL for each 60-mm dish or 2 mL for each 100-mm dish) to collect remaining cells.
7. If some cell clumps are too big, pipet the cell solution up and down several times using
a 5-mL pipette to break the cell clumps into smaller pieces.
8. Aspirate the MEF medium from each MEF culture vessel and replace it with an
appropriate amount of pre-warmed hESC medium (5 mL for each 60-mm dish or 10 mL
for each 100-mm dish).
9. Gently shake the conical tube containing the hESCs to distribute the cell clumps evenly
and add an appropriate amount of hESC suspension into each MEF culture vessel.
Note: The volume of hESC suspension added into each dish depends on the ratio of
splitting (see General Guidelines, above).
10. Move the culture vessels in several quick back-and-forth and side-to-side motions to
disperse the hESCs across the surface of the vessels. Place the culture vessels gently in a
37°C incubator with a humidified atmosphere of 5% CO2.
11. Replace the spent medium daily. hESCs need to be split every 4–10 days based upon
their appearance.
50
Neural Cell Culture and Differentiation
Differentiating hESCs
2. Roll the StemPro® EZPassage™ Disposable Stem Cell Passaging Tool across the entire
vessel in one direction (left to right). Rotate the culture vessel 90 degrees and roll the
tool across the entire dish again.
3. Using a cell scraper, gently detach the cells off the surface of the culture vessel. Gently
transfer the cell clumps into a 50-mL conical tube using a 5-mL pipette.
4. Add 1 mL of pre-warmed hESC EB medium into each well of 6-well plate, 2 mL into
each 60-mm dish, or 3 mL into each 100-mm dish to collect remaining cells and add
them to the 50-mL conical tube containing the hESC.
6. Aspirate the supernatant from the hESC pellet. Gently re-suspend the pellet with an
appropriate amount of EB medium (15 mL for all the cells from one 60-mm dish or
40 mL for all cells from one 100-mm dish).
7. Transfer the cell clumps to an uncoated T-75 flask for a couple of hours. This allows the
fibroblasts to differentially attach to the flask.
8. After a few hours, set the T-75 flask down at a tilted angle to allow the EBs to settle
in one corner of the flask. Aspirate the EB medium and replace it with 40 mL of fresh
EB medium. Transfer the cell clumps to a fresh T-75 flask and incubate them in a 37°C
incubator with a humidified atmosphere of 5% CO2.
9. Feed the EBs with EB medium every day for 4 days. When feeding, set the flask down
at a tilted angle so that the EBs settle in one corner of the flask. Aspirate almost all spent
EB medium, replace it with pre-warmed EB medium, and return flask to the incubator.
Note: Due to DNA release form dead cells, cell clumps may stick together. In this case,
gently pipet the EBs up and down 2–3 times using a 5-mL pipette. This will help you
clean the dead cells off the EB surface. If the EBs attach to flask, use a 5-mL pipette to
blow the attached EBs off the bottom of the flask.
After the EBs float in the neural induction medium for 2 days, they are ready to be
differentiated.
Note: If the EB attach to the flask, use a 5-mL pipette to blow the attached EBs off the
bottom of the flask.
5. Dilute laminin in D-PBS to 20 μg/mL and coat ten 100-mm culture dishes using
2.5–3 mL of laminin for each dish. Incubate the laminin-coated culture dishes in a 37°C
incubator for several hours.
Note: Laminin may form a gel when thawed too rapidly. To avoid this, thaw slowly in
the cold (2°C–8°C). Once thawed, aliquot into polypropylene tubes and store at –5°C to
–20°C. Do not freeze and thaw laminin repeatedly.
6. After incubation, aspirate the laminin and add 10 mL of pre-warmed neural induction
medium into each 100 mm dish.
7. Transfer the EBs from the T-75 flask into a 50-mL tube and centrifuge for 3 minutes at
200 × g.
9. Gently shake the 50-mL tube containing EBs to distribute the EBs evenly and add 1 mL
of EB suspension into each laminin-coated culture dish.
10. Move the culture dishes in several quick back-and-forth and side-to-side motions to
disperse the EBs across the surface of the dishes. Place the dishes gently in a 37°C
incubator with a humidified atmosphere of 5% CO2.
11. Feed the EBs every other day with fresh pre-warmed neural induction medium until
early rosettes form (approximately 2–3 days).
12. To direct the neural precursors to the midbrain fate, feed the differentiating EBs every
other day with neural induction medium containing 100 ng/mL FGF-8b and
200 ng/mL sonic hedgehog (SHH) for 5–6 days.
Note: Plate the EBs at a density of 200–250 per one 100-mm dish. Generally, all EBs
from hESCs cultured in one 100-mm dish can be plated into eight to ten 100-mm dishes.
The variation is from the confluence of hESCs and efficacy of EB formation
Isolating DA Progenitors 1. Label all differentiating colonies containing rosettes using a microscope marker.
2. Using a 200-μL pipette tip pointing to the center of each marked colony, blow off the
cells in rosettes.
3. Use a 10-mL pipette to transfer the detached cell clumps into a 50-mL centrifuge tube.
Note: You can combine the cell clumps from five 100-mm dishes into one 50-mL tube.
52
Neural Cell Culture and Differentiation
5. Aspirate the supernatant and resuspend the cell clumps in 40 mL of neural expansion
medium containing 100 ng/mL FGF-8b and 200 ng/mL SHH.
6. Transfer the cell clumps to a T-75 flask and place the flask in a 37°C incubator with a
humidified atmosphere of 5% CO2.
The rosettes will roll up to form neurospheres after about 1 day in the incubator.
7. Replace half of the neural expansion medium containing 100 ng/mL FGF-8b and
200 ng/mL SHH with fresh medium every other day.
Note: Contaminating non-neural cells tend to attach to the flask. When changing the
medium, set the flask down at a tilted angle to allow the neurospheres to settle in one
corner of the flask. Aspirate half of the neural expansion medium and use a 10-mL
pipette to transfer the neurospheres with the rest of the spent neural expansion medium
to a fresh T-75 flask. Add 20 mL of pre-warmed fresh neural expansion medium to the
flask and incubate in a 37°C incubator with a humidified atmosphere of 5% CO2.
You can perform this procedure several times to purify the neural cells.
DA Neuron Differentiation 1. Coat the surface of the culture vessel (with or without cover slips) with poly-L-ornithine
working solution at 20 μg/mL in distilled water (14 mL for T-75, 7 mL for T-25, 3.5 mL
for 60-mm dish, 2 mL for 35-mm dish) and incubate the vessel overnight at room
temperature.
2. Wash the poly-L-ornithine-coated vessel 4 times with distilled water, and then coat it
with laminin working solution at 10 μg/mL in D-PBS without calcium or magnesium
(14 mL for T-75, 7 mL for T-25, 3.5 mL for 60-mm dish, 2 mL for 35-mm dish). Incubate
the culture vessel for 3 hours at 37°C.
Note: You may coat the culture vessels in advance, replace the laminin solution with
D-PBS without calcium or magnesium, and store them wrapped tightly in Parafilm for
up to 1 week. Make sure that the culture vessels do not dry out.
3. After the neurospheres float in neural expansion medium for 6–8 days, transfer them
into a 15-mL tube and centrifuge for 5 minutes at 200 × g.
5. Gently pipet the cell clumps up and down to break the larger clumps into a single cell
suspension.
6. Centrifuge the cells for 5 minutes at 200 × g and aspirate the supernatant.
9. Aspirate the laminin from the coated culture vessels and plate the dissociated DA
progenitors.
10. Incubate the cells in a 37°C incubator with a humidified atmosphere of 5% CO2 and
replace the spent medium with fresh neural differentiation medium every other day.
11. You can evaluate DA neuron differentiation 3–4 weeks after plating.
Summary
Dopaminergic (DA) neurons are located in the ventral midbrain (VM). The ability
to isolate precursor cells and neurons from the VM provides a powerful means to
characterize their differentiation properties and to study their potential for restoring
dopamine neurons degenerated in Parkinson’s disease (PD). This chapter describes
methods to differentiate precursor cells derived from embryonic ventral mesencephalon
into DA neurons.
Required Materials
54
Neural Cell Culture and Differentiation
Preparing Reagents
Poly-L-Ornithine Stock
Solution Make a 10-mg/mL stock solution of poly-L-ornithine in distilled water. Filter-sterilize
using a 0.22-μm filter and store for up to 12 months at −20°C.
Dissection Buffer For 100 mL of dissection buffer, aseptically mix the following components. The buffer
can be stored at 4°C for 1 week. Add ascorbic acid solution before use.
Component Amount
HBSS 98 mL
D-Glucose 360.3 mg
Penicillin/Streptomycin 2 mL
Preparing Media
Differentiation Medium For 100 mL of differentiation medium, aseptically mix the following components. The
medium can be stored at 4°C for 1 week and add ascorbic acid solution before use.
Component Amount
Neurobasal® Medium 98 mL
L-Glutamine 1 mL
B-27® Supplement 2 mL
FBS 1 mL
Preparing Matrix
3. Incubate the culture vessel at 37°C in a humidified atmosphere of 5% CO2 for at least
2 hours.
6. Add 2 mL of 10 μg/mL laminin solution to a 35-mm dish (0.5 mL for a 4-well plate or
slide, 0.25 mL for a 8-well slide).
7. Incubate the culture vessel at 37°C in a humidified atmosphere of 5% CO2 for at least
2 hours. Store at 4°C until use.
Note: You may coat the plates in advance and store them at 2-8°C, wrapped tightly
with Parafilm®, for up to 4 weeks.
Except for the initial steps of collecting the uterine horns, work under sterile conditions
in a laminar flow hood, or add antibiotics (penicillin/streptomycin at standard
concentrations) to reagents. Perform the steps in a timely manner, and keep the tissue
cooled on ice and immersed in ice-cold buffers throughout the procedure.
Collecting Embryos 1. Using aseptic technique, collect the uterine horns from a time-pregnant rat (staged to
E14 of gestation) or mouse (staged to E13 of gestation).
2. Submerge uterine horns in a 100-mm petri dish containing ice-cold, sterile HBSS, and
carefully rinse 2–3 times with 15 mL ice-cold, sterile HBSS.
3. Transfer the uterine horns to a clean 100-mm petri dish containing dissection buffer.
4. Under a dissection microscope placed in a laminar flow hood, dissect each embryo from
the uterine sac and remove the amniotic membranes.
5. Use a Morian-type perforated spoon to transfer the embryo to a clean sterile petri dish
containing ice-cold dissection buffer.
6. Confirm the gestational age by measuring and recording the crown rump length of the
embryos (10–12 mm for E14 rat or E13 mouse embryos). Exclude any malformed or
otherwise damaged embryos.
56
Neural Cell Culture and Differentiation
2. Hold the tissue with forceps near the forebrain or hindbrain region to avoid damage to
the midbrain region of interest. Carefully dissect and remove the overlying scalp tissue
to isolate the brain.
3. Place the isolated brain in a clean 60-mm petri dish containing dissection buffer on ice.
4. Stabilize the brain with forceps near the forebrain or hindbrain regions, carefully
remove and discard the fore- and hindbrain regions using a scalpel or microscissors.
Make the rostral cut close to the forebrain vesicles and thalamic region, and the caudal
cut at the isthmus region.
2. Use small microscissors or the very tip of a curved scalpel blade to gradually dissect
open the midbrain tube along the dorsal midline.
5. Trim the outermost (most dorsal) areas of the midbrain tube by dissecting away two
thirds of the tissue on each side (approximately lateral/posterior to the sulcus limitans
as an anatomical landmark).
6. Transfer the resulting tissue piece (~0.3 mm × 1.0 mm in dimension) into a conical tube
containing cold dissection buffer kept on ice. Use ~0.2 to 0.5 mL of buffer volume for
each piece of VM tissue.
Dissociating Cells 1. Wash the pieces of VM tissue in cold dissection buffer (e.g., 15 mL of buffer in a 15-mL
conical tube) by letting the tissue pieces sink to the bottom of the conical tube. Aspirate
the medium, and fill the tube with fresh buffer.
2. Aspirate the buffer and add 1 mL StemPro® Accutase® for every 10 pieces of VM tissue.
Incubate the tissues 3-15 minutes at 37°C. Observe the digestion process and determine
the optimal duration by test dissociation and homogenization. Avoid over-digestion,
3. Using fire-polished Pasteur pipets with decreasing diameter, gently dissociate the tissue
pieces by pipetting the tissue up and down for a total of ~20 times. Alternatively, you
may dissociate and homogenize the tissue by first using a pipettor with a 1000-μL tip,
followed with a 200-μL tip. Avoid excessive formation of air bubbles during mechanical
dissociation of VM tissue, as it reduces cell viability.
4. If large pieces of tissue remain in the solution, selectively homogenize the pieces
separately.
5. Optional: Pipet the cell suspension through a cell strainer cap or through a 35- to 70-μm
mesh. To minimize loss of cells from this filtering step, flush the filter membrane with a
small volume of medium after the cell suspension is passed.
6. Centrifuge the cell suspension at 4°C for 3–5 minutes at 200 × g. Aspirate the
supernatant.
7. Resuspend the cells with differentiation medium. Use 200 μL of differentiation medium
for every 10 pieces of midbrain originally isolated.
8. Using aliquot of the cell suspension, determine the cell concentration and viability
by dye exclusion method (Trypan Blue). Use the quantity of live cells counted for
calculating the cell concentration. Cell viability needs to be >80%, and should ideally
range from 95-100%.
DA neurons are among the most fragile cells in the solution. While the cultures will
contain neuronal cell types after relatively harsh treatment, the number of DA neurons
will be low.
3. Culture the cells for 3-10 days, then check for DA neurons by immunocytochemical
staining using antibodies against the neuronal maker β-III-tubulin, and the
dopaminergic markers tyrosine hydroxylase (TH).
58
Neural Cell Culture and Differentiation
Summary
There are numerous protocols available for cryopreserving neural stem cells (NSCs)
derived from human embryonic stem cells; the primary objective of these methods are
the recovery of the cells post-thaw and the retention of their multipotent properties.
This chapter describes a standardized cryopreservation protocol that does not alter the
viability and sublineage differentiation capacity of the preserved cells.
Required Materials
Preparing Media
bFGF 20 ng/mL 1 μg
EGF 20 ng/mL 1 μg
®
StemPro Neural Supplement 2% 1 mL
You may observe a white precipitate when thawing StemPro® Neural Supplement; this
precipitate will disappear when the supplement is completely thawed or dissolved.
Freezing Medium To prepare 10 mL of freezing medium, aseptically mix the following components. For
larger volumes, increase the component amounts proportionally. Filter sterilize the
freezing medium and store at 2–8°C until use.
DMSO 10% 1 mL
Guidelines for
Cryopreserving
Neural Stem Cells • Cryopreserve NSCs when they are 80–90% confluent (2–4 days after seeding).
• Freeze NSCs at a concentration of 2 × 106– 2.4 × 106 viable cells/mL and a volume of
1 mL/vial.
• Use a freezing medium composed of 90% complete StemPro® NSC SFM without the
growth factors (i.e., bFGF and EGF) and 10% DMSO.
• Do not incubate the NSCs in TrypLE™ Select for more than 2 minutes to avoid cell
death.
• Pre-label all cryovials with the following information: cell line, passage number,
concentration, date of freezing, and your initials.
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Neural Cell Culture and Differentiation
Freezing Neural Stem Cells 1. When NSCs are 80–90% confluent (2–4 days after seeding), aspirate the complete
StemPro® NSC SFM from the culture vessel.
2. Wash the cells twice with D-PBS. Aspirate the D-PBS and discard.
3. Add 1 mL of pre-warmed TrypLE™ Select to the culture vessel and incubate at 37°C for
2 minutes.
Note: Do not incubate the NSCs in TrypLE™ Select for more than 2 minutes to avoid cell
death. Neutralize TrypLE™ Select by adding complete StemPro® NSC SFM immediately
after the incubation period (see below).
4. Detach the NSCs from the culture vessel by pipetting off the cells or by tapping the
culture vessel against the heel of your hand.
5. Stop the TrypLE™ Select treatment by adding 5 mL of complete StemPro® NSC SFM.
6. Gently pipet the NSCs up and down to get a single cell suspension and transfer the cell
suspension into a sterile 15-mL conical tube.
7. Centrifuge the NSCs at 200 × g for 5 minutes. Aspirate the supernatant and discard.
8. Resuspend the cell pellet in a minimal volume of pre-warmed complete StemPro® NSC
SFM and remove a sample for counting.
10. Gently aspirate the medium from the conical tube and drop-wise add pre-chilled (4°C)
freezing medium to resuspend the cells at a concentration of 2 × 106– 2.4 × 106 viable
cells/mL.
11. Transfer 1 mL of the NSC suspension in freezing medium into each pre-labeled, pre-
chilled (4°C) cryovial.
12. Transfer the cryovials to the Cryo 1°C Freezing Container and place the container into a
–80°C freezer. This procedure ensures that the cells freeze slowly.
13. The next day, transfer the cells into a liquid nitrogen.
Summary
Required Materials
Rat Brain Cells • Homogenous cell preparation from E18 rat brain tissue as described in Chapter 9,
Isolation, Culture, and Characterization of Cortical and Hippocampal Neurons
(page 40).
Cryopreservation
Freezing Neural Cells 1. Isolate and prepare a suspension of rat brain cells in Neurobasal® medium
supplemented with 2% B-27® as described in Chapter 9, Isolation, Culture, and
Characterization of Cortical and Hippocampal Neurons (page 40).
4. Resuspend the cell pellet in cold Synth-a-Freeze® at a concentration of 2.0 × 106-1.0 × 107
cells/mL.
5. Make 1 mL aliquots of the cells in pre-labeled, pre-chilled cryovials and place the vials
in an isopropanol chamber at 4°C for 10 minutes.
62
Neural Cell Culture and Differentiation
7. Transfer the frozen vials to the vapor phase of liquid nitrogen storage until use is
required.
Cell Recovery
3. Wipe down the vial with ethanol and tap gently on a surface so that all of the medium
collects at the bottom of the tube.
5. Rinse a pipette tip with medium and very gently transfer the cells from the vial to a pre-
rinsed 15-mL tube.
8. Mix the suspension very gently with P-1000 pipette. Avoid creating any air bubbles.
10. Plate ~1 × 105 cells per well in poly-D-lysine coated 48-well plate or an 8-chambered
slide. Bring the cell suspension volume to 500 μL per well by adding complete
Neurobasal®/B-27® medium.
12. Feed the cells every third day by aspirating half of the medium from each well and
replacing it with fresh medium.
Cell Analysis
Summary
The polyanionic dye calcein AM is well-retained within live cells, producing an intense
uniform green fluorescence in live cells (excitation/emission ~495 nm/~515 nm), while
ethidium homodimer-1 (EthD-1) enters cells with damaged membranes to produce a
bright red fluorescence in dead cells (excitation/emission ~495 nm/~635 nm).
Required Materials
Media and Reagents • LIVE/DEAD® Viability/Cytotoxicity Assay Kit (Cat. no. L-3224)
–– Calcein AM
–– Ethidium homodimer-1 (EthD-1)
• Dulbecco’s Phosphate-Buffered Saline (D-PBS) (Cat. no. 14040)
Preparing Reagents
1. Remove the stock solutions provided in the kit from the freezer and allow them to
warm to room temperature.
64
Cell Analysis
3. Combine the reagents by adding 5 μL of the supplied 4-mM calcein AM stock solution
(Component A) to the 10 mL of EthD-1 solution in D-PBS. Vortex the resulting solution
to ensure thorough mixing.
Note: This reagent mixture is suitable for most neural cells. For cells with higher
esterase activity, you might need to start with a lower calcein AM concentration.
For further information, refer to the user manual provided with the LIVE/DEAD®
Viability/Cytotoxicity Assay Kit.
Note: Prepare a freshly coated culture vessel each time before plating cells. There is no
need to rinse the culture vessel before use.
Methods
1. Aspirate the medium supernatant and wash the cells gently with the same volume of
D-PBS prior to the assay to remove or dilute any serum esterase activity.
Microplate reader: Add an aliquot of the cell suspension to each microplate well in a
sufficient volume to cover at least the bottom of each well. Then add an approximately
equal volume of prepared LIVE/DEAD® reagent.
3. Incubate the cells at room temperature for 10–30 minutes. Measure fluorescence using
the appropriate excitation and emission filters.
Determining Viability of
Cells in Suspension with
Flow Cytometry Allow all the reagents to come to room temperature before proceeding.
2. Prepare a 1-mL suspension of cells with 0.1 × 106 to 5 × 106 cells/mL for each assay.
Cells may be in culture medium or buffer.
3. Add 2 μL of a 50-μM calcein AM working solution and 4 μL of the 2-mM EthD-1 stock
to each milliliter of cells. Mix the sample.
4. Incubate the cells for 15–20 minutes at room temperature, protected from light.
5. As soon as possible after the incubation period (within 1–2 hours), analyze the stained
cells by flow cytometry using 488 nm excitation and measuring green fluorescence
emission for calcein (i.e., 530/30 bandpass) and red fluorescence emission for EthD-1
(i.e., 610/20 bandpass).
6. Gate on cells to exclude debris. Using single color-stained cells, perform standard
compensation. The population should separate into two groups: live cells will show
green fluorescence and dead cells will show red fluorescence (Figure 1).
Figure 1 Flow cytometry viability assay using the LIVE/DEAD® Viability/Cytotoxicity Kit. A 1:1 mixture of live
and ethanol-fixed human B cells was stained with calcein AM and EthD-1 following the protocol provided.
Flow cytometry analysis was performed with excitation at 488 nm. The resulting bivariate frequency
distribution shows the clear separation of the green fluorescent (530 nm) live cell population from the red
fluorescent (585 nm) dead cell population.
66
Neural Stem Cell Culture and Differentiation
Summary
After cells are isolated from tissue or differentiated from pluripotent precursors, the
resulting population needs to be characterized to confirm whether the target population
has been obtained. This chapter lists cell-type specific antibody markers commonly
used for immunocytochemical (ICC) and flow cytometric analysis of neural subtypes.
* These are recommended starting concentrations for ICC applications; optimal working
concentrations must be determined empirically.
Summary
Required Materials
Titrating Antibodies
4. Use 1 × 106 cells for each dilution. Smaller numbers of cells ranging from 50,000 to
100,000 may work as well.
5. Centrifuge cells at 300 × g for 5 minutes at 4°C and discard the supernatant.
6. Add 5 μL of antibody from each dilution into separate sample tubes containing cells.
7. Prepare negative controls of cells that have not been stained with antibody, and cells
stained with an isotype control.
9. If primary antibodies are not directly conjugated to fluorescent tags, carry out the
second step incubation with secondary antibody tagged to a fluorescent tag.
68
Cell Analysis
10. Wash with 10 mL of Staining Medium. Discard the supernatant and resuspend the cells
in 0.5 mL of Staining Medium.
Note: Use the same cell number in every experiment. Starting with larger numbers of
cells is preferred since setting up parameters during flow cytometry analysis takes time
and collecting >10,000 events produces more reliable data.
2. Add 5 μL of diluted primary antibody conjugated to a fluorescent tag to the cell pellet.
3. Flick the tube to resuspend the cell pellet. Mix well and incubate on ice for
25-30 minutes.
4. Wash the cells with 10 mL of cold Staining Medium. Centrifuge the cells at 300 × g, 4°C
for 5 minutes.
5. Discard the supernatant and resuspend the cells with 0.5 mL of Staining Medium.
6. Filter the cell suspension through FACS filter tubes before analysis or sorting the cells
by flow cytometry.
Note: For negative controls, prepare cells that have not been stained with antibody, and
cells stained with an isotype control.
3. Flick the tube to resuspend the cell pellet. Mix well and incubate on ice for
25-30 minutes.
4. Wash the cells with 10 mL of cold Staining Medium. Centrifuge the cells at 300 × g, 4°C
for 5 minutes.
6. Mix well and incubate the cells on ice for 25-30 minutes.
7. Wash the cells with 10 mL of cold Staining Medium. Centrifuge the cells at 300 × g, 4°C
for 5 minutes.
8. Discard the supernatant and resuspend cells with 0.5 mL of Staining Medium.
9. Filter the cell suspension through FACS filter tubes before analysis or sorting the cells
by flow cytometry.
Note: For negative controls, prepare cells that have not been stained with antibody, and
cells stained with an isotype control.
70
Cell Analysis
Immunocytochemistry
Summary
Required Materials
Cells • Primary Rat Cortex Neurons (Cat. no. A10840-01) or Primary Rat Hippocampus
Neurons (Cat. no. A10841-01)
Media and Reagents • Neurobasal® Medium (1X), liquid (Cat. no. 21103-049)
• B-27® Serum-Free Supplement (50X), liquid (Cat. no. 17504-044)
• GlutaMAX™-I Supplement (Cat. no. 35050-061)
• Trypan Blue Stain (Cat. no. 15250-061)
• Dulbecco’s Phosphate-Buffered Saline (D-PBS) (1X), liquid (with calcium and
magnesium) (Cat. no. 14040)
• Goat serum (Cat. no. 16210-064)
• MAP2, Mouse Monoclonal Antibody (Cat. no. 13-1500)
• Rabbit anti-GFAP (Glial Fibrillary Acid Protein) (Cat. no. 08-0063)
• Alexa Fluor® 488 goat anti-mouse IgG (Cat. no. A11029)
• Alexa Fluor® 594 goat anti-rabbit IgG (Cat. no. A11037)
• 4’, 6-diamidino-2-phenylindole, dihydrochloride (DAPI) (Cat. no. D1306)
• ProLong® Gold antifade reagent (Cat. no. P36930)
• Paraformaldehyde (4%)
• Triton®-X
Methods
2. Prepare a working solution of the poly-D-lysine stock from step 1 in D-PBS (with
calcium and magnesium) at a concentration of 50 μg/mL.
5. Aspirate the poly-D-lysine solution, and rinse 3 times with nuclease-free water.
Note: Rinse thoroughly, since extra poly-D-lysine can be toxic to the cells.
6. Leave the plates uncovered in the hood until the wells are completely dry. Plates can be
used when dry or can be covered with Parafilm® and stored at 4°C for up to two days.
Maintaining Neuronal
Cultures 1. Thaw cryopreserved primary rat cortex cells according to the instructions provided
with the cells.
2. Plate the cells onto a multi-chambered slide that has been treated with poly-D-lysine.
Seed 1 × 105 cells per chamber in 500 μL of medium.
4. After 24 hours of incubation, aspirate half of the medium from each well and replace
with fresh medium. Return the slide to the incubator.
5. Feed the cells every third day by aspirating half of the medium from each well and
replacing with fresh medium.
Immunocytochemistry
Analysis 1. Before proceeding, prepare a solution of 5% goat serum in D-PBS with calcium and
magnesium. This solution will be used to coat the cells before antibody detection and to
dilute the antibody. Prepare enough solution to completely coat the cells twice.
2. When you are ready to perform the immunocytochemistry procedure, aspirate the
supernatant from each chamber and rinse the cells twice with D-PBS with calcium and
magnesium.
4. Rinse the cells three times with D-PBS with calcium and magnesium.
72
Cell Analysis
5. Permeabilize the cells with 0.3% Triton®-X (diluted in D-PBS with calcium and
magnesium) for 5 minutes at room temperature.
6. Rinse the cells three times with D-PBS with calcium and magnesium.
7. Add enough 5% goat serum solution from step 1 to the cells to coat them, and incubate
for 60 minutes at room temperature.
8. Remove the solution from the wells and coat the cells with primary antibody (mouse
anti-MAP2, 10 μg/mL, and/or rabbit anti-GFAP, 4 μg/mL) diluted in 5% goat serum
solution.
10. Rinse the cells three times with D-PBS with calcium and magnesium.
11. Treat the cells with a secondary antibody (Alexa Fluor® 488 goat-anti mouse (H+L),
10 μg/mL, and/or Alexa Fluor® 594 goat-anti rabbit (H+L), 10 μg/mL) diluted in
5% goat serum solution.
13. Rinse the cells three times with D-PBS with calcium and magnesium.
14. Stain the cells with a DAPI solution (3 ng/mL) for 10 minutes.
15. Mount the cells with ProLong® Gold Antifade Reagent and observe them under the
microscope using filters for FITC, Cy5, and DAPI.
Typical Results Thawed cortical neurons cultured in Neurobasal® Medium supplemented with
B-27® Serum-Free Supplement and GlutaMAX™-I Supplement show a >90% neuron
population with a minimum number of astrocytes when stained with MAP2 antibody.
Within 3–4 days in culture, the neurons display extensive neurite outgrowth that keeps
on increasing as long as they are kept healthy in culture. Results vary if neurons are
cultured in the presence of serum.
Figure 1 Primary rat hippocampus neurons. Immunofluorescence detection of primary neuronal cells
stained with mouse anti-MAP2 antibody (green) and astrocytes stained with rabbit anti-GFAP antibody
(red). Nuclei are stained with DAPI (blue).
Electrophysiology
Summary
Required Materials
Cells • Neural stem cells, cultured on poly-D-lysine coated 96-well plate or other culture
vessel
Preparing Reagents
Fluo-4 AM Loading Solution Fluo-4 AM loading solution consists of 3 μM fluo-4 AM (reconstituted in DMSO) and
0.1% Pluronic® F-127 in Hanks’ Balanced Salt Solution (HBSS). Use the fluo-4 AM
loading solution as soon as possible after preparation to avoid decomposition with
subsequent loss of cell loading capacity.
1. To reconstitute fluo-4 AM, add 44 μL of DMSO to one vial of fluo-4 AM (50 μg) and
vortex thoroughly. You may store the fluo-4 AM reconstituted in DMSO protected from
light, frozen, and desiccated for up to one week.
3. Add 50 μL of the ~860 μM fluo-4 AM/ Pluronic® F‑127 solution to 14.3 mL of HBSS.
74
Cell Analysis
1. Wash the NSCs with 100 μL of Hanks’ Balanced Salt Solution (HBSS).
2. Load the NSCs with 100 μL of fluo-4 AM loading solution per well of a 96-well plate.
You may adjust the volume as appropriate to other culture vessels.
3. Incubate the NSCs in the dark at room temperature for ~60 minutes.
4. Wash the fluo-4-loaded NSCs with 100 μL of HBSS and maintain at room temperature
in the dark until data acquisition
Data Acquisition
1. Place the 96-well plate containing the fluo-4-loaded NSCs in an inverted microscope
(e.g., Nikon T2000) for visual inspection and fluorescent imaging.
2. To acquire and analyze data, define regions of interest around a random series of cells
using your software of choice (e.g., MetaFluor, MDS Analytical Technologies).
Note: The NSCs should display a typical neuronal morphology with dendritic and
axonal processes clearly recognizable by cellular polarity and proportionate size.
3. Identify 50–100 neurons for data acquisition and analysis in each well examined.
4. Excite the NSCs with 488-nm light (e.g., Lambda DG-4 light source) and collect images
from 520-nm emitted light with a CCD or digital camera (e.g., ORCA-ER).
5. Challenge the cells in one well with a neurotransmitter or other ligand. For example,
add 20 μL of 3 mM acetylcholine to achieve a final concentration of 500 μM
acetylcholine in the well.
6. Collect the data using the appropriate software (e.g., MetaFluor, MDS Analytical
Technologies).
Data Analysis
1. Integrate the acquired fluo-4 520-nm emission signal for each region of interest,
normalize to the first ten data points (F/F0) and then plot against time.
2. Set the response criteria. For example, a NSC might be considered responsive to a
given neurotransmitter or ligand if the resulting normalized signal rises more than 10%
within 60 seconds following neurotransmitter addition compared to the baseline signal.
The number of NSCs that exhibit clear changes in intracellular Ca2+ ([Ca2+]i) depends on
the neurotransmitter and differentiation state of the NSCs.
Molecular Characterization
Summary
After cells are isolated from tissue or differentiated from pluripotent precursors, the
resulting population needs to be characterized to confirm whether the target population
has been obtained. The table below lists PCR primers that can be used in quantitative
polymerase chain reactions (qPCR) to measure the expression levels of specific genes
for characterizing neural stem cells (NSCs) and their sublineages.
Target Primer Sequence Tm (°C) Amplicon size (bp) Intron size (bp)
Neural stem cells SOX1-F GCGGAAAGCGTTTTCTTG 53.0
406 No Intron
SOX1-R TAATCTGACTTCTCCTCCC 50.2
76
Molecular Characterization
Summary
A rapid method of analysis for determining the identity of neural stem cells (NSCs) and
their sublineages involves the early detection of differentiation markers tracked at the
RNA level. This protocol follows methodologies described in the PureLink™ RNA Mini
Kit manual for isolating total RNA from neural stem cells (NSCs), followed by cDNA
synthesis using Superscript® III reverse transcriptase. The following protocol gives you
a step-by-step procedure for template preparation required for RT-PCR or qPCR.
Required Materials
Reagents and Equipment • PureLink™ RNA Mini Kit (Cat. no. 12183-018A)
–– RNA Lysis Solution
–– Wash Buffer I
–– Wash Buffer II
–– RNase-free water
–– RNA spin cartridges
–– Collection tubes
–– RNA recovery tubes
• Superscript® III First Strand Synthesis SuperMix (Cat. no. 18080-400)
–– Superscript® III/RNaseOUT™ Enzyme Mix
–– 2X First-strand Reaction Mix
–– Annealing Buffer
–– 50 mM Oligo(dT)20
–– Random hexamers (50 ng/mL)
• β-Mercaptoethanol (Cat. no. 21985-023)
• TrypLE™ Express Stable Trypsin Replacement Enzyme (Cat. no. 12604-013)
• Dulbecco’s Phosphate-Buffered Saline (D-PBS) without Ca2+ and Mg2+ (Cat. no. 14190)
• Ribonuclease H (RNase H) (Cat. no. 18021-071)
• 10X BlueJuice™ Gel Loading Buffer (Cat. no. 10816-015)
• Table-top centrifuge
RNA Isolation
Isolating RNA Important: Perform all steps on ice unless noted otherwise. For all incubations, heat the
thermocyclers in advance. Pre-chill all reagents and thaw all frozen reagents and cells
immediately prior to use. Use RNase-free pipette tips with aerosol barriers.
2. Remove media from T-25 flasks, rinse once with Dulbecco’s phosphate-buffered saline
(D-PBS) and treat cells with 1 mL of pre-warmed TrypLE™ reagent for 10 minutes at
37°C.
3. Harvest the cells and place them into 15-mL centrifuge tubes. Take 100 μL of the sample
and obtain a viable cell count.
4. Centrifuge the cells in a tabletop centrifuge for 7 minutes at 100 × g. Discard the
supernatant.
6. Allow the cell pellet to thaw. Add 0.5 mL of RNA Lysis Solution for each T-25 flask
harvested for the pellet (0.5 mL per 2 × 106–5 × 106 cells). Pipet the cells ~20 times until
the pellet is disrupted.
7. Transfer 0.5 mL of cell lysis solution to 1.5-mL RNase-free microcentrifuge tubes and
centrifuge at room temperature for 2 minutes at 12,000 × g (12,000 rpm).
8. Add 0.5 mL of 70% ethanol to each tube, and vortex the suspension 5–10 times.
9. Apply a 600 μL aliquot of sample to the RNA Spin Cartridge. Centrifuge at room
temperature for 15–30 seconds at 12,000 × g, then discard the flow-through. Continue
applying 600 μL aliquots of the same RNA sample to the spin cartridge until the entire
sample has been processed.
10. Add 700 μL Wash Buffer I to the spin cartridge and centrifuge at room temperature
for 15–30 seconds at 12,000 × g. Discard the flow-through and the tube. Place the spin
cartridge into a clean 2 mL RNA Wash Tube.
11. Add 500 μL Wash Buffer II (containing ethanol) to the spin cartridge and centrifuge at
room temperature for 15–30 seconds at 12,000 × g. Discard the flow-through. Centrifuge
for 1 minute to dry the cartridge.
12. Place the cartridge into an RNA Recovery Tube. Add 40 μL of RNase-free water to the
cartridge, and let it stand for 1 minute. Centrifuge the cartridge at room temperature for
2 minutes at 12,000 × g. Add an additional 40 μL of RNase-free water to the cartridge
and repeat the step. Yield should be about 60–300 μg total RNA.
Note: Always allow time for the RNase-free water to percolate into the cartridge bed.
Do not spin the cartridge immediately because it may result in partial recovery and
alter the yield of RNA. To recover more RNA, add an additional 40 μL of RNase-free
water to the cartridge and repeat the last step for a third time.
78
Molecular Characterization
Determining RNA Quality 1. Measure ratio of absorbance at 260 nm and 280 nm by analyzing 1 μL of the RNA
sample using a NanoDrop™ spectrophotometer. Conduct readings three times, and use
the average as the final value. Wipe down the analysis stage with a lab tissue wetted
with DEPC water before and after measuring each RNA sample. The A260/280 of pure
RNA is ~2.
Note: The yield and quality of the isolated RNA depends on the type and age of the
starting material, in addition to how the material was collected and preserved.
Component Amount
RNA sample 1 µL
™
2X BlueJuice gel loading buffer 1 µL
DEPC-treated water 8 µL
3. Mix the components and load the samples onto individual wells of an agarose gel. Use
10 μL of 0.1 kb and 1 kb molecular weight markers to estimate the molecular weight
size of ribosomal RNA bands. Use 10 μL DEPC water for empty wells. Run samples for
30 minutes, visualize the bands on an UV light box, capture the gel image, and perform
band intensity measurements.
RNA Storage Store RNA samples at -70°C or process it further for cDNA synthesis.
cDNA Preparation
First-Strand cDNA
Synthesis This protocol follows the methodologies described in the instructions for
Superscript® III First Strand Synthesis SuperMix.
1. Mix and briefly centrifuge each component before use. Pre-heat the thermocycler to
65°C.
2. Combine the following components on ice in a 0.2-mL thin-walled PCR tube. Use a
volume containing up to 1 μg of total RNA for the reaction.
Component Amount
Annealing buffer 1 µL
RNA (1 µg) x µL
DEPC-treated water to 8 µL
3. Incubate the reaction in the thermocycler at 65°C for 5 minutes, and then immediately
place on ice for at least 1 minute. Collect the contents of the tube by brief centrifugation.
4. While the tube is on ice, add the following components to the tube:
Component Amount
5. Vortex the sample briefly, and collect the contents by brief centrifugation.
8. Terminate reaction by incubating at 85°C for 5 minutes, then chill the tube on ice.
80
Molecular Characterization
Summary
Quantitative polymerase chain reaction (qPCR) is one of the most accurate and sensitive
methods for studying gene regulation, and can be used to measure the expression levels
of specific genes in neural stem cells (NSCs). These genes can be used to characterize
the NSCs and their respective sublineages.
Here we provide guidelines and a general protocol for performing qPCR using the
Applied Biosystems 7300 Real-Time PCR System and Platinum® SYBR® Green qPCR
SuperMix-UDG with ROX Reference Dye.
Required Materials
Starting Material • cDNA generated from total RNA isolated from neural stem cells (NSCs) (see
Chapter 20, RNA Isolation and cDNA Preparation from Neural Stem Cells, page 77)
Media and Reagents • Platinum® SYBR® Green qPCR SuperMix-UDG (Cat. nos. 11733-038, 11733-046)
• SuperScript® VILO™ cDNA Synthesis Kit (Cat. nos. 11754-050, 11754-250)
• TRIzol® Reagent (Cat. nos. 15596-018, 15596-026)
• Custom primers (www.invitrogen.com/oligos)
Special Tools • Applied Biosystems 7300 Real-Time PCR System or similar instrument
• 0.2-mL microcentrifuge tubes or 96-well or 384-well PCR plates
• Vortex mixer
• Microcentrifuge
Methods
Template Preparation For qPCR, prepare a 1:10 dilution series of cDNA generated from 10 pg–1 μg of
total RNA using the protocol described in Chapter 20, RNA Isolation and cDNA
Preparation from Neural Stem Cells (page 77).
Real-Time PCR Instruments Platinum® SYBR® Green qPCR SuperMix-UDG can be used with a variety of
real‑time instruments, including but not limited to the following Applied Biosystems
instruments: 7300 and 7500 Real-Time PCR Systems; PRISM® 7000, 7700, and 7900HT;
and GeneAmp® 5700. Optimal cycling conditions will vary with different instruments.
Primer Design Primer design is one of the most important parameters when using a SYBR® Green
qPCR detection system. We strongly recommend using a primer design program such
as OligoPerfect™, available at www.invitrogen.com/oligos, or Vector NTI Advance®
software. When designing primers, the amplicon length should be approximately
80–250 bp. Optimal results may require a titration of primer concentrations between
100 nM and 500 nM. A final concentration of 200 nM per primer is effective for most
reactions.
ROX Reference Dye ROX Reference Dye is recommended to normalize the fluorescent reporter signal for
instruments that are compatible with that option. ROX is supplied as a separate tube
in Platinum® SYBR® Green qPCR SuperMix-UDG at a 25 μM concentration. Use the
following table to determine the amount of ROX to use with a particular instrument.
Protocol for qPCR For protocols for specific instruments, visit www.invitrogen.com/qpcr. In this section
we provide a step-by-step protocol for qPCR on the 7300 Real-Time PCR System
(Applied Biosystems) in 20-μL assays.
Melting Curve Analysis: Program the instrument for melting curve analysis to identify
the presence of primer dimers and analyze the specificity of the reaction. A typical
melting curve program is listed below (see your instrument documentation for details):
Note: For the following steps, do not touch the bottom of each tube, and be sure to use
powder-free gloves to handle all reagents and plasticware.
82
Molecular Characterization
2. For each reaction, add the following components to a 0.2-mL microcentrifuge tube or
each well of a PCR plate. Volumes for a single 20-μL reaction are listed. For multiple
reactions, prepare a master mix of common components, add the appropriate volume to
each tube or plate well, and then add the unique reaction components (e.g., template).
For no-template controls, add an equivalent volume of water in lieu of template.
Component Amount
DEPC-treated water to 20 µL
3. Cap or seal the reaction tube/PCR plate, and gently mix. Make sure that all components
are at the bottom of the tube/plate; centrifuge briefly if needed.
Figure 1 qPCR detection of Nestin transcripts in human embryonic stem cell-derived NSCs.
Figure 2 qPCR detection of Sox1 transcripts in human embryonic stem cell-derived NSCs.
Transfection
Summary
The Neon® Transfection System is a benchtop electroporation device that uses the
pipette tip as an electroporation chamber to efficiently transfect mammalian cells
including primary cells and stem cells.
Instructions for using the Neon® Transfection System for transfecting of neural cells
are described below. For detailed instructions on using the Neon® Transfection System,
refer to the manual supplied with the product or download the manual from
www.invitrogen.com. For detailed information on culture conditions for various neural
cell lines, refer to the instructions supplied with the specific cell line you are using.
Required Materials
84
Transfection
Culture Conditions
The following table summarizes the culture conditions for various neural cell lines,
including neural stem cells. For detailed instructions on culturing and passaging these
cells, refer to the to the instructions supplied with the specific cell line you are using.
*For increased proliferation of rat astrocytes, you can supplement complete GIBCO® Astrocyte Medium (D-MEM with 1X N-2 Supplement and
10% OneShot™ FBS) with 20 ng/mL EGF. Adding EGF to human astrocyte cultures can increase proliferation, but may result in morphological or
phenotypic changes.
Preparing Media
KnockOut™ D-MEM/F-12 1X 97 mL
GlutaMAX™-I Supplement 2 mM 1 mL
bFGF 20 ng/mL 2 μg
EGF 20 ng/mL 2 μg
D-MEM 1X 89 mL
N-2 Supplement 1X 1 mL
FBS 10% 10 mL
Transfection Protocol
Use this procedure to transfect plasmid DNA into hNSCs in a 24-well format using the
10-μL Neon® Kit. All amounts and volumes are given on a per well basis.
1. Cultivate the required number of cells in the appropriate growth medium (see table
below) such that the cells are 70–90% confluent on the day of the experiment.
2. On the day of the experiment, harvest and wash cells in phosphate buffered saline
(PBS) without Ca2+ and Mg2+.
3. Resuspend the cell pellet in Resuspension Buffer R (included with Neon® Kits) at the
appropriate final density (see the following table).
4. Prepare 24-well plates by filling the wells with 0.5 mL of the appropriate growth
medium without antibiotics and pre-incubate plates at 37°C in a humidified 5% CO2
incubator. If using other plate formats, adjust the volume accordingly.
5. Turn on the Neon® unit and enter the following electroporation parameters in the
Input window. Alternatively, press the Database button and select the appropriate
transfection protocol (if you have already added the electroporation parameters for
your cell type). For detailed instructions, refer to the manual supplied with the Neon®
unit.
Cell type Cell density Pulse voltage (V) Pulse width (ms) Pulse number Neon® tip
1400 20 2
Human Neural Stem Cells 1 × 107 cells/mL 1600 20 1 10-μL
1700 20 1
1100 30 1
Human Astrocytes 1 × 107 cells/mL 10-μL
1200 40 1
1300 20 2
Rat Fetal Neural Stem Cells 1 × 107 cells/mL 1500 10 3 10-μL
1600 10 3
1400 20 2
Rat Primary Cortical Astrocytes 0.5 × 107 cells/mL 1400 30 1 10-μL
1700 20 1
1300 10 3
Rat Glial Precursor Cells 1 × 107 cells/mL 10-μL
1500 20 1
86
Transfection
6. Fill the Neon® Tube with 3 mL of Buffer E. (Use Buffer E2 if you are using the 100-μL
Neon® Tip.)
7. Insert the Neon® Tube into the Neon® Pipette Station until you hear a click, indicating
that the tube has locked in position.
Note: The quality and concentration of DNA used for electroporation plays an
important role for the transfection efficiency. We strongly recommend using high
quality plasmid purification kits such as PureLink™ HiPure Plasmid DNA Purification
Kits to prepare DNA.
9. Add 1 mL of cells (resuspended in step 3) to the tube containing the plasmid DNA and
gently mix.
11. Press the push-button on the Neon® Pipette to the first stop and immerse the Neon® Tip
into the cell-DNA mixture. Slowly release the push-button on the pipette to aspirate the
cell-DNA mixture into the Neon® Tip.
12. Insert the Neon® Pipette with the sample vertically into the Neon® Tube placed in the
Neon® Pipette Station until you hear a click, indicating that the pipette has locked in
position.
13. Ensure that you have entered the appropriate electroporation parameters and press
Start on the Neon® touchscreen.
The Neon® device delivers the electric pulse according to the parameters entered in step
5 and the touchscreen displays Complete to indicate that electroporation is complete.
14. Remove the Neon® Pipette from the Neon® Pipette Station and immediately transfer the
samples from the Neon® Tip into the prepared culture plate containing the appropriate
pre-warmed complete growth medium without antibiotics.
Discard the Neon® Tip into an appropriate biological hazardous waste container.
16. Gently rock the plate to assure even distribution of the cells. Incubate the plate at 37°C
in a humidified 5% CO2 incubator.
17. Assay the samples to determine the transfection efficiency (e.g., fluorescence
microscopy or functional assay).
Expected Results
Human Neural Stem Cells GIBCO® Human Neural Stem Cells (Cat. no. N7800-100), cultured in StemPro® NSC
SFM complete medium, were transfected with 0.5 μg of a plasmid encoding the
Emerald Green Fluorescent Protein (EGFP) using the Neon® Transfection system with
the parameters listed in the following table. 48 hours post-transfection, the cells were
analyzed by light (Panel A) and fluorescence microscopy (Panel B).
A B
Human Astrocytes GIBCO® Human Astrocytes (Cat. no. N7805-100) were transfected using the Neon®
Transfection Device and 0.5 μg of a plasmid encoding the Emerald Green Fluorescent
Protein (EGFP); 24 hours post-electroporation, the cells were analyzed by light
(Panel A) and fluorescence microscopy (Panel B).
A B
88
Transfection
Rat Fetal Neural Stem Cells GIBCO® Rat Fetal Neural Stem Cells (Cat. no. N7744-100) were transfected using
the Neon® Transfection Device and 0.5 μg of a plasmid encoding the Emerald Green
Fluorescent Protein (EGFP); 24 hours post-electroporation, the cells were analyzed by
light (Panel A) and fluorescence microscopy (Panel B).
A B
A B
Rat Glial Precursor Cells GIBCO® Rat Glial Precursor Cells (Cat. no. N7746-100) were transfected using the
Neon® Transfection Device and 0.5 μg of a plasmid encoding the Emerald Green
Fluorescent Protein (EGFP); 24 hours post-electroporation, the cells were analyzed by
light (Panel A) and fluorescence microscopy (Panel B).
A B
90
Transfection
Troubleshooting
For troubleshooting tips regarding the culture and passaging of your cells, refer to
the manual provided with the cells. For troubleshooting tips regarding the Neon®
Transfection System, see below.
No Neon® Tip is inserted or Make sure that the Neon® Tip is inserted into Neon® Pipette correctly as
Connection failure the Neon® Tip is inserted described. There should be no gap between the tip and the top head of
incorrectly the pipette.
Air bubbles in the Neon® Tip Avoid any air bubbles in the Neon® Tip while aspirating the sample.
Arcing (sparks) High voltage or pulse length
Reduce the voltage or pulse length settings.
settings
• Use high quality plasmid DNA for transfection (use high quality plasmid
purification kits such as PureLink™ HiPure Plasmid DNA Purification
Kits, Cat. no. K2100) to prepare DNA.
• Resuspend the purified DNA in deionized water or TE buffer (10 mM
Tris-HCl, 1 mM EDTA, pH 8.0) at a concentration between 0.5–5 μg/μL.
Poor DNA quality
• Check the purity of the purified DNA preparation by measurement of
the A260/280 ratio. The ratio should be at least 1.8 for electroporation.
• Do not precipitate DNA with ethanol to concentrate DNA. Concentrated
DNA by ethanol precipitation shows poor transfection efficiency and
Low cell survival cell viability due to salt contamination.
rate
• Avoid severe conditions during cell harvesting especially high speed
centrifugation and pipette cells gently.
Cells are stressed or • Avoid using over confluent cells or cells at high densities as this may
damaged affect the cell survival after electroporation.
• After electroporation, immediately plate the cells into prewarm culture
medium without antibiotics.
Do not use the same Neon® Tip for electroporation for more than 2 times
Multiple use of the same
because the repeated application of electric pulses reduce the tip quality
Neon® Tip
and impair their physical integrity.
Poor plasmid DNA quality • Use high quality plasmid DNA for transfection.
or the plasmid DNA is low • Start with 0.5 μg plasmid DNA per sample.
Mycoplasma contaminated Test cells for Mycoplasma contamination. Start a new culture from a fresh
cells stock.
Inconsistent cell confluency Always use cells with low passage number and harvest cells with
or passage number comparable confluency levels.
Nonreproducible • Do not use the same Neon® Tip for more than 2 times because the
transfection repeated application of electric pulses reduce the tip quality and impair
Multiple use of the same their physical integrity.
efficiency Neon® Tip or the same
• Do not use the same Neon® Tube for more than 10 times.
Neon® Tube
• Always use a new Neon® Tip and Neon® Tube for different plasmid DNA
samples to avoid any cross-contamination.
Summary
Astrocytes are by far the most numerous cell type in the central nervous system (CNS)
and have critical roles in adult CNS homeostasis. They provide biochemical and
nutritional support of neurons and endothelial cells which form the blood-brain barrier,
perform the vast majority of synaptic glutamate uptake, and maintain extracellular
potassium levels (Rothstein et al., 1996; Rothstein et al., 1994). Although there are few
known differences between cortical and hippocampal astrocytes, it has been reported
that astrocytes from different regions of the brain show a differential sensitivity to
ischemic injury (Xu et al., 2001; Zhao & Flavin, 2000).
Required Materials
For transfecting siRNA • Silencer® Select siRNAs (see www.invitrogen.com for ordering information)
• Lipofectamine™ RNAiMAX Transfection Reagent (Cat. no. 13778-075 or 13778-150)
92
Transfection
2. On ice, prepare a stock solution of Geltrex™ diluted 1:1 in D-MEM. Store in aliquots at
–20°C until needed.
3. Dilute the stock solution 1:100 in D-MEM and coat the bottom of each culture vessel
(200 μL of Geltrex™ per cm2 of culture vessel).
4. Incubate the culture vessel at 37°C for 1 hour. Dishes coated with Geltrex™ can be used
immediately or stored at 4°C for up to a week, sealed with Parafilm®. Do not allow
dishes to dry. When you are ready to add cells, aspirate the Geltrex™ solution and rinse
the plates once with D-PBS with Ca2+ and Mg2+ before adding the cell solution.
Aseptically mix the following components for preparing 100 mL of complete GIBCO®
Astrocyte Medium. Complete Astrocyte Medium is stable for 2 weeks when stored at
4°C protected from light.
D-MEM 89 mL 445 mL
N-2 Supplement 1 mL 5 mL
FBS 10 mL 50 mL
1. Warm Complete Astrocyte Medium and StemPro® Accutase® Cell Dissociation Reagent
in a 37°C water bath before use.
2. Transfer conditioned medium from the cells to a new tube; this will be used to stop the
enzyme reaction in step 6.
3. Wash cells once with 1X D-PBS without calcium, magnesium, or phenol red.
5. Incubate for 5–10 minutes at 37°C. Rock the cells every ~5 minutes and check under a
microscope for detachment and dissociation toward single cells.
6. When the cells have detached, add an equal volume (1:1) of conditioned medium (from
Step 2) to slow the Accutase® activity.
8. Rinse culture vessels with complete medium and add it to the tube.
13. To replate human astrocytes, remove a Geltrex™-coated plate from 4°C storage and tip
slightly to aspirate the Geltrex™ solution. Rinse the plate once with D-PBS with calcium
and magnesium. Do not allow the plate to dry out.
14. Immediately seed the astrocytes at the desired concentration (we recommend ≥2 × 104
cells/cm2).
15. Incubate the cells in an incubator at 37°C in a humidified atmosphere (90%) of 5% CO2
in air. Change the medium every 2–3 days with fresh Complete Astrocyte Medium.
94
Transfection
Transfecting Plasmid DNA into Human Astrocytes Using Lipofectamine™ LTX Reagent
Use this procedure to transfect plasmid DNA into GIBCO® Human Astrocytes using the
Lipofectamine™ LTX Reagent in a 24-well format (for other formats, see Scaling Up or
Down Transfections, below). All amounts and volumes are given on a per well basis.
1. The day before transfection, prepare Human Astrocytes that have recovered from
cryopreservation and have reached 80% confluency. Use StemPro® Accutase® to detach
the cells and count the cells. Plate 5 × 104 cells per well in 0.5 mL of complete growth
medium. Cell density should be 80–90% confluent on the day of transfection.
2. For each well of cells to be transfected, dilute 0.5 μg of DNA into 100 μL of Opti-MEM® I
Reduced Serum Medium without serum.
3. Using PLUS™ Reagent: Mix PLUS™ Reagent gently before use, then add 0.5 μL PLUS™
Reagent (a 1:1 ratio to DNA) directly to the diluted DNA. Mix gently and incubate for
5–15 minutes at room temperature.
4. For each well of cells, dilute 1.5–3.0 μL of Lipofectamine™ LTX into the above diluted
DNA solution, mix gently and incubate for 25 minutes at room temperature to form
DNA-Lipofectamine™ LTX complexes.
5. Remove growth medium from cells and replace with 0.5 mL of complete growth
medium. Add 100 μL of the DNA-Lipofectamine™ LTX complexes directly to each well
containing cells and mix gently by rocking the plate back and forth.
Scaling Up or Down
Transfections To transfect Human Astrocytes in different tissue culture formats, vary the amounts
of Lipofectamine™ LTX Reagent, DNA, cells, medium and PLUS™ Reagent used in
proportion to the relative surface area, as shown in the table (amounts given on a per
well basis).
Volume Volume of
Culture Surface area Cells per Lipofectamine™ PLUS™
of plating dilution DNA
vessel per well* well LTX Reagent Reagent
medium medium†
Prepare siRNAs
1. Resuspend the Silencer® Select siRNAs with nuclease-free water. A convenient stock
concentration is 100 μM, which can be diluted to meet downstream experimental needs.
4. From the working stock dilute siRNAs in 20 μL of Opti-MEM® I per well to achieve a
final concentration of 30 nM or your desired concentration (1 nM–100 nM) in tubes or
plates. When applicable, make master mixes for replicates to minimize variability (at
least one well overage).
For example with a 10 μM siRNA stock, mix 0.39 μL of siRNA (3.9 pmols) + 19.61 μL of
Opti-MEM®-I per well or 1.56 μL of siRNA + 78.44 μL of Opti-MEM®-I for the master mix.
5. Plate siRNAs in a Geltrex™-coated plate using the proper method for coating as
provided by the manufacturer
Prepare cells Culture cells according to manufacturer’s cell protocol. Cells were in culture for about
a week. On the day of transfection, harvest cells according to the protocol. Count and
dilute cells to the proper density. We recommend performing an initial optimization
experiment to determine your optimal cell density. Based on our optimization, we
found 4,000 cells per well to be an optimal cell density.
Transfect cells We recommend performing an initial optimization experiment to determine the optimal
amount of transfection agent to add that balances good knockdown and low toxicity.
Based on our optimizations, we found 0.15 μL per well of Lipofectamine™ RNAiMAX to
be the best condition.
3. After the incubation, add 80 μL of human astrocytes that have been diluted to the
proper density to each well. The final volume per well should be 110 μL per well.
4. Place the plate in a 37°C incubator under normal cell culture conditions. Remove the
cells and assay for the expression levels of the gene of interest at the desired time point
(typically 24–48 hours post-transfection).
96
Transfection
References
Rothstein, J.D., Dykes-Hoberg, M., Pardo, C.A., Bristol, L.A., Jin, L., Kuncl, R.W., et al.
1996. Knockout of glutamate transporters reveals a major role for astroglial transport in
excitotoxicity and clearance of glutamate. Neuron 16:675–686.
Rothstein, J.D., Martin, L., Levey, A.I., Dykes-Hoberg, M., Jin, L., et al. 1994. Localization
of neuronal and glial glutamate transporters. Neuron 13:713–725.
Xu, L., Sapolsky, R.M., and Giffard, R.G. 2001. Differential sensitivity of murine
astrocytes and neurons from different brain regions to injury. Exp Neurol. 169:416–424.
Zhao, G., and Flavin, M. P. 2000. Differential sensitivity of rat hippocampal and cortical
astrocytes to oxygen-glucose deprivation injury. Neurosci Lett. 285:177–180.
Summary
Required Materials
98
Using Neural Cells for Cell Therapy
Preparing Reagents
Preparing
6-hydroxydopamine
(6‑OHDA) Solution Make a 2-mg/mL solution of 6-hydroxydopamine (6-OHDA) in saline. Store protected
from light up to 12 months at −20°C.
Preparing the Animal 1. Weigh a rat and place it into an isoflurane chamber and apply oxygen and isoflurane
until the animal is deeply anesthetized.
2. Position the rat in a stereotactic frame and fix the plastic tube connected to the
anesthesia machine to the nose of the rat using surgical tape. Maintain isoflurane at
~1.5% with an oxygen flow of 2−3 liters/minute.
3. Shave the top of the rat’s head with an electric razor. Clean the skin with betadine and
70% ethanol.
4. Perform a midline incision with a scalpel and identify the bregma at the intersection of
the coronal and the sagittal sutures.
5. Adjust the incisor bar in the rat until the heights of lambda and bregma skull points are
equal.
6. Calculate the stereotactic coordinates for injection. For a MFB lesion, coordinates are in
reference to the bregma: Anteroposterior (A/P) −2.2 mm; mediolateral (M/L) 1.5 mm.
Administering 6-OHDA 1. Fill a 10-μL Hamilton syringe with 5 μL 6-OHDA solution. Attach the syringe to the
holder on the stereotactic frame.
2. Lower the needle of the Hamilton syringe so that it is 8 mm from the dura.
4. Leave the needle in place for 5 minutes and withdraw the tip slowly.
5. Close scalp margins with sutures or staples. Remove the rat from the stereotactic frame
and place it in its home cage. Put food on the floor of the cage and monitor the animal’s
weight for 3 days after surgery.
Evaluating the Behavior At 10−14 days after injection of 6-OHDA, the rats that exhibit at least 210 contralateral
rotations over 30 minutes when challenged with the dopamine receptor agonist
apomorphine (0.2 mg/kg, i.p.), or 630 ipsilateral rotations over 90 minutes when
challenged with the DA-releasing substance amphetamine (5 mg/kg, i.p.) are suitable
for future study.
Appendix
Overview
Life Technologies provides you with all of your neural cell culture needs through its
GIBCO® Cell Culture Media and offers products including reagents, media, sera, and
growth factors to support the growth of a range of neural cell lines. All cell culture
media products available from Life Technologies are tested for contamination and
guaranteed for their quality, safety, consistency, and regulatory compliance. For more
information on Invitrogen and GIBCO® products, refer to www.invitrogen.com.
Cells
100
Appendix
Media
Supplements
Reagents
102
Appendix
Accesory Products
104
Appendix
Books
Developmental Biology, 9th edition, edited by Scott F. Gilbert Sinauer Associates, 2010.
Neural Stem Cells, 2nd edition, edited by Lesile P. Weiner, Humana Press, 2008.
Neural Development and Stem Cells, 2nd edition, edited by Mahendra S. Rao, Humana
Press, 2006.
Protocols for neural cell culture, 4th edition, edited by Laurie C. Doering, Humana
Press, 2010.
Protocols for neural cell culture, 3rd edition, edited by Sergey Fedoroff and Arleen
Richardson, Humana Press, 2001.
Journals
Developmental Neurobiology,
www.onlinelibrary.wiley.com/journal/10.1002/%28ISSN%291097-4695
Organizations
Government Sites
Websites
EMA, http://www.ema.europa.eu/ema
FDA, www.fda.gov/BiologicsBloodVaccines/CellularGeneTherapyProducts
106
Appendix
Technical Support
Web Resources
Contact Us
For more information or technical assistance, call, write, fax, or e-mail. Additional
international offices are listed on our website (www.invitrogen.com).
SDS
Certificate of Analysis
The Certificate of Analysis provides detailed quality control and product qualification
information for each product. Certificates of Analysis are available on our website. Go
to www.invitrogen.com/support and search for the Certificate of Analysis by product
lot number, which is printed on the product packaging (tube, pouch, or box).
The trademarks mentioned herein are the property of Life Technologies Corporation or
their respective owners.
For research use only. Not intended for any animal or human therapeutic or
diagnostic use.
108
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