Magnetofection TM

the new gene transfection technology

MagnetofectionTM is a novel, simple and highly efficient

method to transfect cells in culture.
Table of contents

Technology 2

1.1. Description 2

1.2. Magnetofection™ Reagents 3

1.3. Nucleic Acids Dose Response and Transfection Kinetic 3

1.4. Application 4

Utensils for Magnetofection™ 11

2.1. MagnetoFACTOR plates 11

Avalaible Kits 12

3.1. Kits Contents 12

3.2. Contact 12

Protocols 13

4.1. General Considerations 13

4.2. General Protocol 13

4.3. Magnetofection in 6-, 12-, 24- or 96-well Plate Formats, T-75 Culture Flasks 14
4.4. PolyMAG 16

4.5. CombiMAG 17
4.5.1. CombiMAG – Example Protocol for Fugene (Roche) 18
4.5.2. CombiMAG – Example Protocols for other Cationic Lipid Reagents 19
4.6. Magnetofection™ - Suspension Cells 19

4.7. Magnetofection™ - siRNA 20

4.7.1. siRNA – PolyMAG Protocol 21
4.7.2. siRNA – CombiMAG Protocol 22
4.8. Magnetofection™ - Viruses – CombiMAG Protocol 24

4.9. High Throughput Optimizations 26

4.10. Magnetofection™ - Optimization Protocol in 96-well Format 26

4.11. Troubleshooting 28

References 30
Technology

1.1. Description

Magnetofection™ is a novel, simple and highly efficient method to transfect cells in

culture. It exploits magnetic force exerted upon gene vectors associated with
magnetic particles to draw the vectors towards, possibly even into, the target cells. In
this manner, the full vector dose applied gets concentrated on the cells within a few
minutes so that 100% of the cells get in contact with a significant vector dose. This
has several important consequences:

• Greatly improved transfection rates in terms of percentage of cells transfected

compared to standard transfection.

• Up to several thousand fold increased levels of transgene expression compared to

standard transfections upon short-term incubation.

• High transfection rates and transgene expression levels are achievable with
extremely low vector doses, which allows to save expensive transfection reagents.

• Extremely short process time. A few minutes of incubation of cells with gene
vectors are sufficient to generate high transfection efficiency, compared to several
hours with standard procedures.

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Technology

1.2. Magnetofection™ Reagents

As the manufacturer of the Magnetofection™ technology, chemicell offers two types

of ready-to-use Magnetofection™ reagents.

PolyMAG is a universally applicable magnetic particle preparation for high efficiency

nucleic acid delivery. It is mixed in a one-step procedure with the nucleic acid to be
transfected and has been used successfully with plasmid DNA, antisense
oligonucleotides and siRNAs.

CombiMAG is a magnetic particle preparation designed to be combined with any

commercially available transfection reagent such as polycations and lipids and can be
associated with plasmid DNA, antisense oligonucleotides, siRNAs or viruses.
It allows you to create your own magnetic gene vector based on your favourite
transfection reagent.

Purchaser Notification

The Magnetofection™ reagents and all of its components are developed, designed, intended and sold
for research use only. They are not to be used for human diagnostic or any drug intended.

Magnetofection™ are registered trademark

1.3. Nuclec Acids Dose Response and Transfection Kinetics

paramagnetic 200
response (expression level)

vehicle plus
response (expression level)

300
magnetic field 150

200
100
paramagnetic
100
vehicle no
magnetic field 50

0 0
0 50 100 150 200 250 standard 0 0.05 0.1 0.15

process time (min) gene transfer dose (µg DNA)

Transfection kinetics: NIH 3T3 cells Dose response profile in NIH 3T3
were incubated with GenePorter™ cells using Lipofectamine™
(Gene Therapy Systems) ± (Invitrogen) ± CombiMAG with and
CombiMAG with and without without positioning on the
positioning on the Magneto- MagnetoFACTOR plate for 15 min.
FACTOR plate for the indicated Luciferase expression was assayed
time spans. Luciferase expression after 24 hours.
was assayed after 24 hours.

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1.4. Application

Magnetofection™ is generally applicable for adherent cells and has been tested with
a variety of immortalized cell lines and primary cells listed below. If a particular cell
type or cell line is not listed this does not mean that Magnetofection™ would not work.
Also for the cells listed, some reagents have not been tested so far, as indicated by
“n.d.” (not determined).

The CombiMAG reagent can be combined with any polycationic and lipidic
transfection reagent, and also with adenoviral and retroviral vectors. In some cases,
references are made in the footnotes to very successful combinations with
commercially available reagents that have been tested so far.

Type of nucleic acid / virus PolyMAG CombiMAG

Plasmid DNA √ √
Antisense Oligonucleotides √ √
siRNA √ √
Adenovirus n.d. √
Retrovirus n.d. √

Primary human umbilical vein endothelial cells positioned on the

MagnetoFACTOR plate were incubated for 15 min with a Cy3 fluorescence-
labeled antisense-oligonucleotide complexed with Effectene™ (Qiagen; left)
or Effectene™ + CombiMAG (right).

(Data kindly provided by F. Kroetz. Ludwig-Maximilians University Munich)

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Technology

Adherent Cells
Cell line Cell line Source PolyMAG CombiMAG* Ref. No.
9.030, 9.024,
9.072, 9.012,
HeLa cervix carcinoma human + + 9.111, 9.003,
9.005, 9.016,

9.013, 9.064,
HEK 293 kidney human + + 9.054,

BHK kidney hamster n.d. + -

9.036, 9.002,
CHO ovarian hamster + + 9.005, 9.006,

9.003, 9.072,
NIH 3T3 fibroblasts mouse + + 9.091, 9.002,
9.005, 9.004,

16HB140 airway epithelium human + n.d. 9.010,

EJ28 bladder cancer human + n.d. -

HBL-100 breast human n.d. + -
9.014, 9.040,
MCF7 breast adenocarcinoma human + + 9.041, 9.115,

MCS7 breast adenocarcinoma human + n.d. -

HCT 116 colon adenocarcinoma human n.d. + -
U373 astrocytoma human n.d. + 9.056,

U87 glioblastoma human n.d. + 9.056,

U251 glioblastoma human n.d. + 9.056,

T98G glioblastoma human n.d. + 9.056,

YK6-1 glioblastoma human n.d. + 9.056,

YH-13 glioblastoma human n.d. + 9.056,

A549 alveolar basal epithelial human + + 9.075, 9.101,

A172 glioblastoma human n.d. + 9.056, 9.074,

LN229 glioblastoma human + + 9.074,

LN18 glioblastoma human + + 9.074,

SW480 colon adenocarcinoma human n.d. + -

LoVo colon adenocarcinoma human n.d. + -
HCT15 colon adenocarcinoma human n.d. + -
CEMx174 hybrid T/B-lymphocytes human n.d. + 9.060,

CHO ovarian human + + -

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A431 epidermoid carcinoma human n.d. + -

HT-1080 fibrosarcoma human + n.d. -
HFF foreskin fibroblast human + + -
Hep G2 hepatocellular carcinoma human + + 9.012, 9.048,

HaCat keratinocytes human n.d. + -

293 T kidney human + + 9.030,

293 T-17 kidney human n.d. + 9.030,

SK-MES-1 lung carcinoma human + n.d. -

MRC 5 lung, embryonic human + + -
MeWo melanoma human n.d. + -
NYGM cerebral glioblastoma human n.d. + 9.056,

SK-MEL-28 melanoma human n.d. + -

MEP mammalian epithelial human n.d. + 9.047,

MOLT-4 acute lymphoblastic leukemia human n.d. + 9.044,

SHSY-5Y neuroblastoma human n.d. + 9.111, 9.050,

SV40 transformed fibroblasts human + n.d. 9.027,

A549 Non-small cell lung carcinoma human n.d. + 9.057, 9.101

SaOS-2 osteo sarcoma human n.d. + -

BTK-143 osteo sarcoma human n.d. + -
SaOS-2 osteo sarcoma human n.d. + -
BEAS-2B lung epithelial cells human + n.d. 9.012

181RDB pancreatic human n.d. + -

HN12 Head, neck carcinoma human n.d. + 9.028

PC-3 prostate carcinoma human n.d. + -

H9 lymphoblastoid human n.d. + 9.077

9.058, 9.059,
H295R adrenocortical carcinoma human n.d. + 9.037

HOS osteosarcoma human n.d. + 9.077

salivary gland,
HSG human n.d. + -
submandibular.
He99 lung cancer human + + 9.100

HMEC-1 microvascular endothelial human + n.d. 9.038, 9.092

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Technology

lung adenocarcinoma
NCI-H441 human + + 9.073
epithelial
9.065, 9.095,
NCI-H292 mucoepidermoid carcinoma human + n.d. 9.112

NCI-H82 small cell lung cancer human + n.d. -

SK-N-BE2 neuroblastoma human + n.d. 9.031

MDAMB231 breast adenocarcinoma human + n.d. 9.035

ECV-304 urinary bladder carcinoma human n.d. + -

11.5dpcXY gonad explants mouse n.d. + 9.109

oral adenosquamous
Cal27 mouse n.d. + 9.028
carcinoma
CT-26 colon carcinoma mouse + n.d. 9.003, 9.005

C127 murine mammary tumor mouse n.d. + 9.042

F9 embryonal carcinoma mouse + n.d. -

L929 fibroblast, connective tissue mouse + n.d. 9.016

MEF fibroblast mouse + n.d. 9.039, 9.098

HT-22 hippocampal neuroblast mouse n.d. + -

mIEnd Mesenteric lymph node mouse n.d. + 9.085

mIC-(cl2) intestine mouse n.d. + -

B16F10 melanoma mouse + n.d. 9.076, 9.005

N2a neuroblastoma mouse + n.d. 9.063, 9.086

NS20Y neuroblastoma mouse n.d. + -

bTC-tet B-cell line mouse + + 9.020

Hepa1c1c7 murine hepatoma mouse + n.d. 9.084

SVEC murine cell line mouse n.d. + 9.028

OP9 bone marrow-derived stromal mouse n.d. + 9.047

SM 10 trophoblast mouse n.d. + -

STC-1 enteroendocrine I cells mouse + + 9.033

A7r5 aortic smooth muscle rat n.d. + 9.094

PC12 adrenal pheochromocytoma rat + n.d. -

PC6-3 daughter cell line of PC12 rat + n.d. -
C6 glioma rat n.d. + -

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Technology

H4IIE hepatoma rat n.d. + 9.062

L6 skeletal muscle cell rat + n.d. -

RIE1-α5 intestinal rat + n.d. 9.088

9.015, 9.028,
HNSCC Head, neck carcinoma rat n.d. + 9.051,

RV vein rat + n.d. -

COS-7 kidney monkey + + 9.030

CV-1 kidney monkey + n.d. -

COS-1 kidney monkey n.d. + -
9.045, 9.046,
Vero kidney monkey n.d. + 9.016

B11 (UV) resistant ovary hamster n.d. + 9.028

V79 lung fibroblasts hamster + n.d. -

PT-11 kidney bovine n.d. + -
MDCK kidney canine + n.d. 9.067

CRFK kidney cat n.d. + -

VSa13 bone (chondrocyte-like) fish + n.d. -
AM-C6SC8 kidney pig n.d. + -
- fibroblasts xenopus + n.d. -

EMC mesenthelial. Cells (epicard) n.d. + -

Suspension Cells
Cell line Cell type Source PolyMAG CombiMAG* Ref. No.

THP-1 acute myeloid leukemia human n.d. + -

Jurkat acute T-cell lymphoma human n.d. + 9.044, 9.073

BL-41 B-cell lymphoma human + n.d. -

K562 chronic myeloid lymphoma human n.d. + 9.002

P815 mastocytoma mouse n.d. + -

U937 histiocytic lymphoma human + + -

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Technology

Primary Cells
Cell type Source PolyMAG CombiMAG* Ref. No.

endothelial cells (cord blood) human + n.d. 9.054

aortic endothelial cells human + n.d. 9.028, 9.032

9.029, 9.034, 9.092, 9.113,

epithelial (HUVEC) human + + 9.114, 9.003, 9.007, 9.008,
9.021, 9.029, 9.034, 9.005, 9.004

fibroblasts human + + -
fibroblasts, diploid human + + -
gastric gland human + + -
adherent gastric cells human n.d. + 9.069

gastric myofibroblasts human + n.d. 9.043

glioma human + n.d. -

keratinocytes human n.d. + 9.005

LNCaP human n.d. + -

lymphocytes human + + 9.005, 9.102

mammal epithelium human n.d. + -

nasal airway epithelium human + + 9.005

pancreatic tumor human + n.d. -

peripheral blood lymphocytes human + + 9.005, 9.102

stroma cells (endometrium) human n.d. + -

T-cells human + + 9.102, 9.089

chondrocytes human + + 9.107, 9.022, 9.024

trophoblastic cells human n.d. + -

fibroblasts (MEF) mouse n.d. + 9.018, 9.053

neurons mouse + + 9.071

vagal afferent neurons mouse n.d. + 9.071

adherent gastric cells mouse n.d. + 9.061

peripheral blood lymphocytes mouse n.d. + -

myoblasts mouse n.d. + 9.053

T-cells mouse + n.d. -

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Technology

9.017, 9.070, 9.050, 9.052,

hippocampal neurons rat + + 9.093, 9.090, 9.105, 9.104

aortic smooth muscle cells rat n.d. + 9.094

cardiomyocyte rat n.d. + 9.055

hepatocytes rat n.d. + -

epithelial (HUVEC) rat + + 9.028, 9.054

cortical neurons rat n.d. + 9.052, 9.063, 9.097

aortic endothelial cells bovine + n.d. -

carotid artery smooth muscle bovine + n.d. -
chromaffine cells bovine n.d. + -
lens bovine + n.d. -
airway epithelium pig n.d. + -
chondrocytes pig + + -
fibrochondrocytes pig + + -
pSM, smooth muscle pig n.d. + -

* Corresponds to successfully transfected by CombiMAG in association with different commercial

transfection reagents such as FuGENE™ (Roche), Lipofectamine™, METAFECTENE™ (Biontex
GmbH) and DMRIE-C™ (Invitrogen); adenovirus; Effectene™ (Qiagen)

n.d. = not determined

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 Utensils for Magnetofection™

2.1. MagnetoFACTOR plates

Apart from suitable magnetic nanoparticles, Magnetofection™ requires appropriate

magnetic fields. These are provided by the MagnetoFACTOR plate, especially
designed for Magnetofection™.

The MagnetoFACTOR-96 plate its special geometry not only produces strong
magnetic fields under each well of 96-well plates but is also applicable for other plate
formats T-75 culture flasks, 6- and 12-well plates. In the larger plate formats, the
MagnetoFACTOR plate will produce a pattern of higher and lower densities of
transfected cells according to the geometry of the magnetic field lines. The
MagnetoFACTOR-24 plate is special designed for the 24-well format.

The MagnetoFACTOR plates are compatible for well plates of the most manufacturer.

MagnetoFACTOR-96 plate MagnetoFACTOR-24 plate

SPECIAL OFFER

As an introductory offer you will receive one vial each of PolyMAG and CombiMAG
sufficient for 200 µg of DNA each for free in combination with the purchase of the
MagnetoFACTOR plate. This will enable you to optimize your Magnetofection™ in up
to 3000 transfection experiments (96-well format).

@ [email protected]

chemicell GmbH • Magnetofection™ 2.4 11

Avalaible Kits
3.1. Kits Contents

Product Size Number of

Price
Description (sufficient for Transfection
Number n µg DNA)
EUR / USD
(96-well format)
9001 PolyMAG-100 100 1000 95 / 125
9002 PolyMAG-500 500 5000 365 / 480
9003 PolyMAG-1000 1000 10000 665 / 875

9004 CombiMAG-100 100 500 50 / 65

9005 CombiMAG-500 500 2500 180 / 235
9006 CombiMAG-1000 1000 5000 350 / 460

9007 MagnetoFACTOR-96 plate - - 350 / 460

9009 MagnetoFACTOR-24 plate - - 350 / 460

9008-96 SPECIAL OFFER: 350 / 460

MagnetoFACTOR-96 plate - -
PolyMAG-200 200 2000
CombiMAG-200 200 1000
9008-24 SPECIAL OFFER: 350 / 460
MagnetoFACTOR-24 plate - -
PolyMAG-200 200 2000
CombiMAG-200 200 1000
Storage:
All components of the Magnetofection™ kit should be stored at room temperatur (20-25°C).
After first use store the kit at +4°C.

• Do not freeze the magnetic nanoparticles

• Do not add anything to the stock solution of magnetic nanoparticles
• Shipping Conditions: Room Temperatur

3.2. Contact

chemicell GmbH
Eresburgstrasse 22-23
12103 Berlin
Germany
Tel.: +49-30-2141481
@ [email protected]
Fax.: +49-30-21913737
e-mail: [email protected]
Internet: www.chemicell.com

chemicell GmbH • Magnetofection™ 2.4 12

 Protocols

4.1. General Considerations

The instructions given below represent sample protocols that were applied
successfully with a variety of cell lines. Please note that optimal conditions do vary
from cell line to cell line. Therefore, the amount of DNA or RNA used and the ratios of
the individual components may have to be adjusted to achieve best results. The
following recommendations can be used as guidelines to achieve good transfection
with minimal incubation times.

4.2. General Protocol

Adherent cells are seeded such that they reach 60-80% confluency at the time of
Magnetofection™.

For suspension cells, use the specific protocol given in section 4.6. Immediately
preceding transfection, the medium can be replaced with fresh medium (optionally
without serum) if necessary.

The suggested cell number for adherent and suspension cells is given below.

Recommended DNA amount, PolyMAG volume and transfection volume.

Tissue Culture Cell Number DNA Quantity PolyMAG or Transfection

Dishes per well (µg) CombiMAG (µL) Volume*

96-well 0.5 - 2 x 104 0.1 - 0.5 0.1 - 0.5 200 µL

24-well 0.5 - 1 x 105 0.5 - 2 0.5 - 2 500 µL

12-well 1 - 2 x 105 2-4 2-4 1 mL

6-well 1 - 4 x 105 2-6 2-6 2 mL

60 mm dish 5 - 10 x 105 6-8 6-8 5 mL

90-100 mm dish 1 - 2 x 106 8 - 12 8 - 12 10 mL

T-75 flask 2 - 5 x 106 15 - 25 15 - 25 15 - 20 mL

*Total Transfection volume = culture medium + PolyMAG or CombiMAG complex

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 Protocols

The following protocols (section 4.4, 4.5, 4.5.1, 4.5.2 and 4.6) can be used to produce
stably transduced cells except that 48 hours post transfection. Cells are transferred to
fresh medium containing the appropriate antibiotics for selection. It is important to
wait at least 48 hours before exposing the transduced cells to selection media.

Vectors are prepared in medium without serum and supplement or in

physiological saline, because serum may interfere with transfection complex
assembly. The serum and supplement-free transfection complex is added to the
cells that are covered with complete medium. Therefore, the addition of the
transfection complex will result in the dilution of supplements such as serum,
antibiotics or other additives of your standard culture medium. Although a medium
changes after Magnetofection™ is not required for most cell types, it may be
necessary for cells that are sensitive to serum/supplement concentration.
Alternatively, the cells may be kept in serum-free medium during Magnetofection™.
In this case, a medium change will be required after Magnetofection™.

4.3. Magnetofection™ in 6-, 12- , 24- or 96-well Plate Formats, T-75 Culture Flasks

As a rule of thumb, a DNA amount of 50 ng to 300 ng per square centimeter culture

dish will yield good results. However, it is emphasized that every cell line requires
optimization with respect to DNA amount and vector formulation.

The easiest way to generate the complexes is to provide the required amount of
magnetic particles in a microcentrifuge tube, add the required amount of DNA which
has been diluted with serum-free medium (e.g. DMEM). After 15 min incubation, add
the magnetic particles / DNA complex to the cells. Position the 6-well culture plate on
the MagnetoFACTOR plate for up to 10-20 min, subsequent perform a medium
change (optional).

6-well culture dish:

A useful DNA amount for the 6-well format is 2 µg up to 6 µg, whereas the ratio of
magnetic particles / DNA is 1:1. To 1.8 mL cells per well add 200 µL magnetic
particles / DNA complexes.

12-well culture dish:

A useful DNA amount for the 12-well format is 2 µg up to 4 µg, whereas the ratio of
magnetic particles / DNA is 1:1. To 0.8 mL cells per well add 200 µL magnetic
particles / DNA complexes.

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 Protocols

24-well culture dish:

A useful DNA amount for the 24-well format is 0.5 µg up to 2 µg, whereas the ratio of
magnetic particles / DNA is 1:1. To 0.3 mL cells per well add 200 µL magnetic
particles / DNA complexes.

96-well culture dish:

A useful DNA amount for the 96-well format is 0.1 µg up to 0.5 µg, whereas the ratio
of magnetic particles / DNA is 1:1. To 0.15 mL cells per well add 50 µL magnetic
particles / DNA complexes.

T-75 culture flask:

A useful DNA amount for the T-75 culture flask format is 15 µg up to 25 µg, whereas
the ratio of magnetic particles / DNA is 1:1. To 14 mL (19 mL) cells add 1 mL
magnetic particles / DNA complexes.

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 Protocols

4.4. PolyMAG

Plate the adherent cells the day before transfection or suspension cells just before
transfection in the appropriate tissue culture dish and volume of culture medium as
recommended in table section 4.2.
The protocol is as simple as follows: Use 1 µL of PolyMAG per µg of DNA.

1. Add the required amount of PolyMAG (according to the DNA amount) to a

microcentrifuge tube or to a U-bottom well of 96-well plate. If required and for
amount less than 1 µL PolyMAG in your protocol, predilute PolyMAG with
deionized water.

Note: Vortex the PolyMAG before used.

2. Dilute required amount of DNA to 200 µL with serum- and supplement-free

medium (such as DMEM).

3. Add the 200 µL DNA solution to PolyMAG and mix immediately by vigorous
pipetting.

4. After 15 minutes of incubation, add the 200 µL of complexes to the cells.

Note: The total transfection volumes per well (culture medium + PolyMAG complex)
are suggested in the table above.

5. Place the cell culture plate upon the MagnetoFACTOR plate and incubate under
standard cell conditions for 10 to 20 minutes.

6. Remove the MagnetoFACTOR plate.

6a.Optionally perform a medium change.

7. Cultivate cells under standard conditions until evaluation of transgene expression.

STANDARD TRANSFECTION MAGNETOFECTION™

Confluent primary human Keratinocytes transfected with PolyMAG

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 Protocols

4.5. CombiMAG

A number of suppliers sell efficient transfection reagents. All of these can be made a
magnetofectin by simple mixing with CombiMAG, usually resulting in strong
improvements of these reagents efficiencies.

There are two strategies of using CombiMAG:

• One is to prepare a standard DNA complex with a commercial transfection reagent

according to the instructions of the manufacturer, followed by mixing with
CombiMAG.

• The second strategy is to first mix DNA and CombiMAG followed by mixing with the
transfection reagent.

Also in this case, the instructions of the manufacturer are used with the only
exception that instead of DNA alone, a mixture of DNA and CombiMAG is added to
the transfection reagent.

Depending on the transfection reagent, the mixing order of components may

influence the final transfection efficiency. It is recommended to use 1 - 2 µL of
CombiMAG per µg of DNA in initial experiments.

However, depending on the cell line to be transfected and the commercial

transfection reagent used, the optimal composition may be found above or below this
ratio.

Note: If required in your setup, predilute CombiMAG with ddwater.

CT-26 colon carcinoma cells were CT-26 colon carcinoma cells were
transfected for 15 min with a GFP transfected for 15 min with a GFP
reporter plasmid complexed with reporter plasmid complexed with
DMRIE-C (Invitrogen) DMRIE-C (Invitrogen) + CombiMAG
on a magnetic plate

(Data kindly provided by Ch. Plank. Technical University Munich)

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 Protocols

4.5.1 CombiMAG - Example Protocol for Fugene (Roche)

Plate 0.5 x 105 adherent cells per well the day before transfection or suspension cells
just before transfection in a 24-well culture plate.

1. Add 2.4 µL of Fugene to 97.6 µL serum- and supplement-free medium (such

as DMEM) and vortex vigorously for 2 seconds.

2. Dilute 0.8 µg of DNA to 100 µL with serum- and supplement-free medium

(such as DMEM).

3. Mix the DNA solution with the Fugene dilution by pipetting and incubate for 15
minutes at room temperature.

4. Add the resulting 200 µL of DNA complex to 1.6 µL of CombiMAG and mix
immediately by vigorous pipetting. Vortex the CombiMAG before used.

5. After further 15 minutes of incubation add the DNA / Fugene / CombiMAG

complex to the cells.

6. Place the cell culture plate upon the MagnetoFACTOR plate and incubate under
standard cell culture conditions for 10 to 20 minutes.

7. Remove the magnetic plate and cultivate cells under standard conditions until
evaluation of transgene expression.

8. Optionally perform a medium change.

9. Depending on the commercial transfection reagent used, this protocol may have
to be adapted.

Note: If required in your setup, predilute CombiMAG with ddwater.

Human Keratinocytes Porcine Chondrocytes

Transfection of primary cells
Vector Fugene™ (Roche) +CombiMAG. 15 min transfection on MagnetoFACTOR plate
(Data kindly provided by Ch. Plank. Technical University Munich)

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 Protocols

4.5.2 CombiMAG - Example Protocols for other Cationic Lipid Reagents

The same steps as for Fugene are carried out. The primary DNA complex is
prepared similar to the instructions of the manufacturer.

For example, in step 1, 3.2 µL of Lipofectamine + 96.8 µL medium or 4.0 µL

GenePorter + 96 µL medium are used instead of Fugene. The rest of the protocol
stays the same.

However, the user is reminded that the alternative mixing order (first mixing DNA and
CombiMAG followed by mixing with lipid) may be advantageous.

Similarly, the polycationic reagents ExGen500 or Superfect (Qiagen) can be used

instead of the lipids.

4.6. Magnetofection™ – Suspension Cells

1. The composition and preparation of PolyMAG / DNA or CombiMAG / transfection

reagent are performed exactly as described above (section 4.4 and 4.5).
2. While PolyMAG / DNA or CombiMAG / transfection reagent incubate for complex
formulation, dilute the cells to be transfected to 5 x 105 - 1 x 106 / mL in medium
(with or without serum- or supplement; depending on cell type and sensitivity of
cells towards serum-free conditions) and perform one of the following three
options to sediment the cells at the bottom of the culture dish in order to promote
the contact with the magnetic nanoparticles.

Option 1: Seed the cells on polylysine-coated plates and use the protocol for
adherent cells.
Option 2: Briefly, centrifuge the cells (2 minutes) to pellet them and use the
protocol for adherent cells.
Option 3: Mix cell suspension with 30 µL of CombiMAG reagent per mL of cell
suspension.
- Incubate for 10 - 15 minutes.
- Distribute cells to your tissue culture dish placed upon the magnetic
plate (volume of culture medium containing cells depends on the
culture dish size; see suggested transfection volume in table section
4.2.
- Incubate for 15 minutes

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 Protocols

3. Add the resulting complex of PolyMAG / DNA or CombiMAG / transfection reagent

to the cells while keeping the cell culture plate on the MagnetoFACTOR plate.

4. Continue to incubate for 15 minutes.

5. Carefully remove the medium supernatant from the cells and replace with fresh
complete medium while the culture plate remains positioned on the
MagnetoFACTOR plate.
Be careful not to aspirate the magnetically sedimented cells.

6. Remove culture plate from MagnetoFACTOR plate.

7. Continue to cultivate cells as desired until evaluation of transgene expression.

4.7. Magnetofection™ - siRNA

RNA interference is a powerful technique to shut down gene expression in cells and
organisms. This silencing effect constitutes a very helpful tool to study gene function
and is a promising approach for new therapeutic treatments. Short RNA duplexes
(siRNA: small interfering RNA, shRNA: small hairpin RNA and dsRNA: double strand
RNA) are extremely selective by interacting and inducing the degradation of their
specific mRNA targets and thereby inhibit the resulting protein production.
PolyMAG introduces the siRNA duplexes in a variety of cells with a very high
efficiency leading to exceptional knockdown effects with low doses of siRNA.

The protocol is very straightforward. Please refer to the tables below for specific
amount of the respective compounds and transfection volume.

For instance use 2 µL of PolyMAG for 20 nM siRNA final concentration in a 6-well

plate (transfection volume 2 mL)

chemicell GmbH • Magnetofection™ 2.4 20

 Protocols

Example dilution procedure of siRNA (1 µM stock solution*) and PolyMAG / CombiMAG :

Culture vessel 96-well 24-well 12-well 6-well

Dilution serum-free medium 100 µL 100 µL 100 µL 200 µL

ng siRNA / ng siRNA / ng siRNA / ng siRNA /

Final siRNA concentration µL PolyMAG µL PolyMAG µL PolyMAG µL PolyMAG
or CombiMAG or CombiMAG or CombiMAG or CombiMAG
2 nM 5.4 / 0.5 19.5 / 1.0 27 / 2.0 54 / 2.0
5 nM 19.5 / 0.5 39.75 / 1.0 67.5 / 2.0 135 / 2.0
10 nM 27 / 0.5 67.5 / 1.0 135 / 2.0 270 / 2.0
20 nM 54 / 0.5 135 / 1.0 270 / 2.0 540 / 2.0
50 nM 135 / 0.5 337.5 / 1.0 675 / 2.0 1350 / 2.0
75 nM 202.5 / 1.0 506.25 / 2.0 1012.5 / 2.0 2025 / 2.0
*ng of siRNA was calculated on the basis of a MW = 13 500 g/mol

4.7.1 siRNA – PolyMAG Protocol

1. Plate the cells the day before transfection or just before transfection in your
appropriate tissue culture dish and volume of culture medium as suggested (see
table section 4.2).

2. Dilute the siRNA to 100 µL (or 200 µL) with culture medium without serum and
supplement (such as DMEM) (see table for siRNA dilution procedure).

3. Vortex the PolyMAG tube before each use. If required, PolyMAG can be diluted
only with deionized water.
Don’t dilute PolyMAG with serum or supplement-free serum.

4. Add directly the appropriate volume/amount of PolyMAG to 100 µL (or 200 µL) of
the diluted siRNA solution and mix immediately 4 - 5 times by vigorous pipetting.

5. Incubate the formed siRNA / PolyMAG complex 15 minutes at room temperature.

6. Add 100 µL (or 200 µL) of the binary complex drop by drop onto the cells.

Note: For some cells, serum-free condition for the first 3 hours of incubation might
lead to better gene silencing. However, in most assays, siRNA delivery has been
realized in culture medium with serum.

chemicell GmbH • Magnetofection™ 2.4 21

 Protocols

7. Place the cell culture plate upon the MagnetoFACTOR plate and incubate under
standard cell culture conditions for 10-20 minutes.

8. Remove the MagnetoFACTOR plate.

9. Cultivate the cells under standard conditions until evaluation of the gene
silencing. Depending on the siRNA amount, the gene target and the cell type
assays can be monitored 24 to 96 hours post-transfection.
We recommend 48 hours and 72 hours for RNA and protein knockdown
analyses, respectively.

Note: Optionally a medium change can be performed 8-24 h after the

transfection if your cells are sensitive to serum/supplement concentration.

4.7.2 siRNA – CombiMAG Protocol

For instance:

• Use 2 µL of CombiMAG for 20 nM siRNA final concentration in a 6-well plate

(Transfection Volume 2 mL)

1. Plate the cells the day before transfection or just before transfection in your
appropriate tissue culture dish and volume of culture medium as suggested (see
table section 4.2).

2. Prepare the binary siRNA / Cationic Lipid Reagent complex similar to the
instructions of the manufacturer. The final volume should be 100 µl (or 200 µl) with
culture medium without serum and supplement (such as DMEM) (see table for
siRNA dilution procedure).

3. Vortex the CombiMAG tube before each use. If required, CombiMAG can be
diluted only with deionized water.
Don’t dilute CombiMAG with serum or supplement-free serum.

4. Add directly the appropriate volume/amount of CombiMAG to 100 µL (or 200 µL) to
the siRNA / Cationic Lipid Reagent complex and mix immediately 4 - 5 times by
vigorous pipetting.

5. Incubate the formed siRNA / Cationic Lipid Reagent / CombiMAG complex 15

minutes at room temperature.

6. Add 100 µL (or 200 µL) of the ternary complex drop by drop onto the cells.

chemicell GmbH • Magnetofection™ 2.4 22

 Protocols
Note: For some cells, serum-free condition for the first 3 hours of incubation
might lead to better gene silencing. However, in most assays, siRNA delivery has
been realized in culture medium with serum.

7. Place the cell culture plate upon the MagnetoFACTOR plate and incubate under
standard cell culture conditions for 10-20 minutes.

8. Remove the MagnetoFACTOR plate.

9. Cultivate the cells under standard conditions until evaluation of the gene
silencing. Depending on the siRNA amount, the gene target and the cell type
assays can be monitored 24 to 96 hours post-transfection. We recommend 48
hours and 72 hours for RNA and protein knockdown analyses, respectively.

Note: Optionally a medium change can be performed 8-24 h after the

transfection if your cells are sensitive to serum / supplement concentration.

Cell culture conditions:

Best results are achieved when cells are 60 - 80 % confluent at the time of the
transfection. If necessary, you can wash the culture medium containing the
transfection mixture after 8-24 hours and replace it by fresh medium.

siRNA concentration:

We often observed good siRNA effects by concentrations from 10 nM to 50 nM.

However the efficiency may depend on the cell line, the target (half life, expression
level…) and the siRNA used. Consequently, we suggest to start by testing a range
of siRNA concentrations in order to obtain the best experimental conditions.

Magnetofection™ Standard transection Untreated cells

Magnetofection of siRNA. Synthetic siRNA directed against eGFP was purchased from MWG Biotech, Ebersberg, Germany. The RNA was mixed with PEI-
coated magnetic particles and additional free linear PEI (25 kDa, Polysciences, Warrington, PA, USA) in DMEM. After 30 min incubation, the mixture was
diluted 51.2-fold with DMEM containing 10% FCS to result in a final siRNA concentration of 65 nM. Aliquots of this mixture (150 µL) were added to HT1080
cells (5000 cells/well in a 96-well plate) which had previously been stably transduced with a retroviral vector coding for eGFP. The culture plate was placed on
a magnetic plate during the first 15 min of incubation. A medium change was performed after 24 h. The figure shows the cells 65 h after transfection,
documenting an efficient knock down of eGFP expression.
(Data kindly provided by Ch. Plank. Technical University Munich)

chemicell GmbH • Magnetofection™ 2.4 23

 Protocols

4.8. Magnetofection™ - Viruses – CombiMAG Protocol

Viral infection is highly cell surface receptor-dependent. For instance, adenoviruses

are dependent on cells to express CAR (Coxsackie’s and adenovirus receptor) and
HIV on cells to express CD4.

Unfortunately, many important and interesting target tissues for fundamental

research and gene therapy are non-permissive to viral gene delivery (tumor tissues
and apical surface of lung epithelium may express variable, little or none of the
required receptors).

• The association of viral vectors with CombiMAG is sufficient to force infection of

non-permissive cells as shown with adenovirus.

• Magnetofection™ also increases retroviral infectious capacity.

NIH 3T3 cells (lacking the coxsackie

and adenovirus receptor, CAR) were
transfected with a recombinant
adenovirus (coding for LacZ) mixed with
CombiMAG in the presence and in the
absence of permanent magnets
positioned under the culture plates.

chemicell GmbH • Magnetofection™ 2.4 24

 Protocols

1. Cells should be plated in the same manner as required for standard viral gene
delivery. For example, the confluency can be high for adenoviral vectors but must
be low for retroviral vectors, which require cell division for infection. Cells must be
plated the day prior transfection.

2. Provide a suitable amount (see examples below) of CombiMAG in a tube large

enough to contain the volume of virus preparation added in step 3.

3. Add your virus preparation (e.g. retroviral supernatant or purified adenovirus

diluted in HBS, PBS or cell culture medium) to the tube(s) containing CombiMAG
and mix immediately by pipetting or gentle vortexing. Thereafter, incubate 15
minutes at room temperature.

4. The ratios virus / CombiMAG should be adjusted according to the viral titers and
cell types used. For optimization, we suggest as a starting point to use 1.5 µL,
3 µL, 6 µL and 12 µL of CombiMAG with a fixed quantity of virus preparation /
supernatant.

5. Add the mixture prepared in step 3 to the cells in duplicate or triplicate.

6. Place the cell culture plate upon the magnetic plate and incubate under standard
cell culture conditions for 10-20 minutes.

7. Remove the MagnetoFACTOR plate. Optionally perform a medium change.

8. Cultivate the cells under standard conditions until evaluation of transgene

expression.

9. Depending on the viral vector type, the quantity of virus and the cell types used,
this protocol would have to be adjusted.

Cell Type Virus CombiMAG

K562 Adenovirus (200 MOI) 6 µL

Human PBL Adenovirus (500 MOI) 3 – 6 µL

NIH-3T3 Adenovirus (200 MOI) 3 – 6 µL

NIH-3T3 Retrovirus (1-5 x 103 X gal CFU/ml) 3 – 6 µL

chemicell GmbH • Magnetofection™ 2.4 25

 Protocols

4.9. High Throughput Optimizations

The ratios of PolyMAG or CombiMAG to DNA can be varied by doubling or

multiplying the volumes of the reagents used.

Similarly, the reagents can be pre-diluted in deionized water and aliquots of the
resulting dilutions are incubated with DNA or pre-formed DNA complexes,
respectively, such as described above.

Finally, the assembled magnetofectins can be serially diluted to very low

concentrations.

4.10. Magnetofection™ - Optimization Protocol in 96-well Format

We recommend to optimize the transfection conditions in order to get the best results
of Magnetofection™. Several parameters can be optimized:

• Ratio of PolyMAG / CombiMAG to nucleic acid or virus

• Amount of nucleic acid

• Cell density

• Incubation time

For adherent cells, seed the cells at the desired density in a 96-well plate the day prior
or at least several hours prior transfection in a total of 150 µL medium per well.

1. In four tubes, dilute 7.2 µg DNA with 352.8 µL serum- and supplement-free
medium (e.g. DMEM)

2. Add 3.6 µL, 7.2 µL, 10.8 µL and 14.4 µL of PolyMAG (in case of DNA) or
CombiMAG (in case of DNA-transfection reagent complex) reagent in well A1, A4,
A7 and A10 of a 96-well plate.

3. Add the 352.8 µL DNA solution from step 1 to well A1, A4, A7, A10 containing
PolyMAG and mix well by pipetting. Incubate for 15 min at room temperature.

chemicell GmbH • Magnetofection™ 2.4 26

 Protocols

4. (Optional) Perform a medium change for the cells to be transfected. Remove

medium the cells have been seeded in and replace with 150 µL fresh medium (with
or without serum or supplements).

5. In the meantime add 180 µL serum- and supplement-free medium (e.g. DMEM)
to the residual wells of column 1, 4, 7 and 10 of the 96-well plate (B1-H1, B4-H4,
B7-H7, B10-H10).

6. After the incubation in step 3 transfer 180 µL from well A1, A4, A7, A10 to B1, B4,
B7, B10, mix by pipetting, transfer 180 µL from B1, B4, B7, B10 to C1, C4, C7,
C10, mix by pipetting and so on down to H1, H4, H7, H10.

7. Transfer 50 µL each in duplicate from column 1, 4, 7 and 10 to the columns of the

cell culture plate where the cells to be transfected have been seeded
(column 2/3 , 5/6 , 8/9 , 11/12).

8. Place the culture plate on the MagnetoFACTOR plate and incubate under cell
culture conditions for 10-20 min.

9. Remove MagnetoFACTOR plate.

10. (Optional) Perform a medium change, particularly if the transfection has been
carried out in serum-free medium.

11. Continue to culture cells as desired.

chemicell GmbH • Magnetofection™ 2.4 27

 Protocols

4.11. Troubleshooting

Low Transfection Efficiency

▪ Inappropriate buffer composition:

Serum-free buffer or medium has to be used for the formation of the
PolyMAG/DNA- or CombiMAG/DNA complex, otherwise proteins from the serum
will bind to PolyMAG or CombiMAG. Once the PolyMAG/DNA-
or CombiMAG/DNA complex is formed it can be applied to cells in the presence
of serum.

▪ Suboptimal ratio of PolyMAG or CombiMAG to nucleic acid or virus:

Determine the optimal ratio of PolyMAG or CombiMAG to DNA by using the
optimization protocol for 96-well plate (section 4.10).

▪ Correct handling of the MagnetoFACTOR plate:

Use the MagnetoFACTOR plate with the magnets facing up. After addition
of the PolyMAG/DNA- or CombiMAG/DNA complex to the cells, position the cell
culture plate on the MagnetoFACTOR plate.

▪ Positive control:
Perform a positive control transfection experiment with a well-characterized
reporter gene (e,g. GFP, Luciferase).

▪ Mycoplasma contamination:
Mycoplasma contamination alters transfection efficiency.

▪ Cell condition:
Cells that have been in culture for a long time may become resistant to
transfection. Use freshly thawed cells that have been passaged at least once.

chemicell GmbH • Magnetofection™ 2.4 28

 Protocols

Cellular Toxicity

▪ Cell density (% confluence) was not optimal at the time of transfection:

Adherent cells are seeded such that they reach 60-80 % confluency at the time of
Magnetofection™. If the cell density is too low, increased toxicity may be
observed. For suspension cells it is necessary that the cells are immobilized on
the well bottom (section 4.6).

▪ Suboptimal amount of DNA (section 4.10):

Using Magnetofection™, approximately 5 x less DNA compared to Lipofection is
necessary.

▪ Purity of transfecting molecule:

Check the purity of the molecule of interest to be delivered (lipopolysaccharides
which are endotoxins will cause cell death).

▪ Amounts of PolyMAG or CombiMAG:

Higher amounts of PolyMAG or CombiMAG may be cell toxic and can additional
reduce the transfection efficiency.

▪ Incubation time on the MagnetoFACTOR plate:

Longer incubation times (e.g. 4 hours) with PolyMAG/DNA- or CombiMAG/DNA
complex plus application of an magnetic field can cause toxic effects.

▪ Incubation time after Magnetofection™:

Reduce the incubation time of complexes with the cells. Transfection medium can
be replaced by fresh medium after 4 hours.

chemicell GmbH • Magnetofection™ 2.4 29

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