Food Frontiers - 2020 - Kamiloglu - Guidelines For Cell Viability Assays

Received: 30 June 2020 Revised: 19 August 2020 Accepted: 27 August 2020
DOI: 10.1002/fft2.44
GUIDELINE
Guidelines for cell viability assays
Senem Kamiloglu1 Gulce Sari2 Tugba Ozdal3 Esra Capanoglu4
1
Mevsim Gida Sanayi ve Soguk Depo Ticaret
A.S. (MVSM Foods), Bursa, Turkey Abstract
2
Department of Gastroenterology and Recently, the interest in the application of cell viability assays has been increasing in
Hepatology, Erasmus University Medical
various fields. Cell viability assays may be broadly classified as (a) dye exclusion assays,
Center, Rotterdam, the Netherlands
3
Department of Food Engineering, Faculty of
(b) colorimetric assays, (c) fluorometric assays, (d) luminometric assays, and (e) flow
Engineering, Istanbul Okan University, Tuzla, cytometric assays. Dye exclusion assays include trypan blue, eosin, congo red, and
Turkey
erythrosine B stain assays, whereas 3-[4,5-dimethylthiazol-2-yl]-2,5 diphenyl tetra-
4
Department of Food Engineering, Faculty of
Chemical and Metallurgical Engineering, zolium bromide (MTT), 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-
Istanbul Technical University, Maslak, Turkey (4-sulfophenyl)-2H-tetrazolium (MTS), 2,3-bis-(2-methoxy-4-nitro-5-sulfophenyl)-
2H-tetrazolium-5-carboxanilide (XTT), 2-(4-iodophenyl)-3-(4-nitrophenyl)-5-(2,4-
Correspondence
Esra Capanoglu, Department of Food Engi- disulfophenyl)-2H tetrazolium, monosodium salt (WST-1), 2-(2-methoxy-4-
neering, Faculty of Chemical and Metallurgical
nitrophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfophenyl)-2H-tetrazolium, monosodium
Engineering, Istanbul Technical University,
34469 Maslak, Istanbul, Turkey. salt (WST-8), lactate dehydrogenase (LDH), sulforhodamine B (SRB), neutral red
Email: [email protected]
uptake (NRU), and crystal violet stain (CVS) assays are among the colorimetric assays.
Similarly, resazurin and 5-carboxyfluorescein diacetate acetoxymethyl ester (5-CFDA-
AM) assays are based on fluorometric measurements, whereas luminometric assays
comprise adenosine triphosphate and real-time viability assays. Major flow cytometric
assays include membrane asymmetry, membrane permeability, and mitochondria
assays. In this guideline, the mechanisms and the practice of assessment of the most
common cell viability assays applied in research labs are discussed in detail. An ideal
cell viability assay should be safe, rapid, reliable, efficient, and time- and cost-effective,
and should not interfere with the test compound. Overall, it can be concluded that
more than one cell viability assay should be applied in order to obtain reliable results.
KEYWORDS
annexin V staining, ATP assay, MTT assay, resazurin assay, trypan blue stain assay
1 INTRODUCTION of cells (Stoddart, 2011). Cell viability assays are essentially used for
screening the response of the cells against a drug or a chemical agent. In
Cell viability is defined as the number of healthy cells in a sample. The particular, pharmaceutical industry widely uses viability assays to eval-
measurement of cell viability plays an important role for all forms of cell uate the influence of developed agents on the cells. Researchers apply
culture. Sometimes it is the main purpose of the experiment as in tox- various types of assays in order to screen the outcome of a developed
icity assays, or it can be used to correlate cell behavior to the number therapeutics that often target cancer cells (Adan, Kiraz, & Baran, 2016).
This is an open access article under the terms of the Creative Commons Attribution License, which permits use, distribution and reproduction in any medium, provided
the original work is properly cited.
© 2020 The Authors. Food Frontiers published by John Wiley & Sons Australia, Ltd and Nanchang University, Northwest University, Jiangsu University, Zhejiang Univer-
sity, Fujian Agriculture and Forestry University
332 wileyonlinelibrary.com/journal/fft2 Food Frontiers. 2020;1:332–349.

KAMILOGLU ET AL . 333
There are several types of assays that can be used to deter-

mine the number of viable cells. These assays are based on various
functions of cells including enzyme activity, cell membrane perme-
ability, cell adherence, adenosine triphosphate (ATP) production,
co-enzyme production, and nucleotide uptake activity (Thangaraj,
2016). Although there are different classifications, cell viability assays
may be broadly categorized as (a) dye exclusion assays, (b) colorimetric
assays, (c) fluorometric assays, (d) luminometric assays, and (e) flow
cytometric assays. Dye exclusion assays are the simplest methods
that are based on utilization of different dyes such as trypan blue,
eosin, congo red, and erythrosine B, which are excluded by the liv-
ing cells, but not by dead cells. For these assays, although staining
procedure is quite straightforward, experimental procedure may be
time-consuming in case of large sample sizes. Colorimetric assays are
F I G U R E 1 Determination of cell viability with trypan blue assay
based on the measurement of a biochemical marker to determine
(modified from Allevi Protocols, 2020)
the metabolic activity of the cells. In these assays, the colorimetric
measurement of cell viability is carried out spectrophotometri-
cally. 3-[4,5-Dimethylthiazol-2-yl]-2,5 diphenyl tetrazolium bromide line, the mechanisms and the practice of assessment of the most com-
(MTT), 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2- mon cell viability assays applied in research labs are discussed in detail.
(4-sulfophenyl)-2H-tetrazolium (MTS), 2,3-bis-(2-methoxy-4-nitro-5-
sulfophenyl)-2H-tetrazolium-5-carboxanilide (XTT), 2-(4-iodophenyl)-
3-(4-nitrophenyl)-5-(2,4-disulfophenyl)-2H tetrazolium, monosodium 2 DYE EXCLUSION ASSAYS
salt (WST-1), 2-(2-methoxy-4-nitrophenyl)-3-(4-nitrophenyl)-5-(2,4-
disulfophenyl)-2H-tetrazolium, monosodium salt (WST-8), lactate 2.1 Trypan blue stain assay
dehydrogenase (LDH), sulforhodamine B (SRB), neutral red uptake
(NRU), and crystal violet stain (CVS) assays are among the most widely Trypan blue stain assay has initially been developed in 1975 to measure
applied colorimetric assays. These assays are simple and economical, viable cell count and is still used as a confirmatory test for measuring
and can be applied to both cell suspensions and adherent cells. Fluoro- changes in viable cell number caused by a drug or toxin (Tolnai, 1975).
metric assays including resazurin and 5-carboxyfluorescein diacetate Trypan blue stain, a large negatively charged molecule, is one of the
acetoxymethyl ester (5-CFDA-AM) assays may be performed with a simplest assays that are used to determine the number of viable cells in
fluorometer, fluorescence microplate reader, fluorescence microscope, a cell suspension (Stone, Johnston, & Schins, 2009). The principle of this
or flow cytometer. These assays are advantageous over dye exclusion assay is that living cells have intact cell membranes that exclude the try-
and colorimetric assays as they are more sensitive. In luminometric pan blue stain, whereas dead cells do not. Cell suspension is mixed with
assays, a persistent and stable glow-type signal is produced following the trypan blue stain and examined visually under light microscopy to
the addition of reagent. These methods comprise ATP and real-time determine whether cells include or exclude the stain. A viable cell will
viability assays (Aslantürk, 2018). Flow cytometry allows simultaneous have a clear cytoplasm, whereas a nonviable cell will have a blue cyto-
measurement of the changes in cell morphology by forward and side plasm (Strober, 2015) (Figure 1).
light scatter, which makes this technology uniquely suited to measur-
ing the complex progression of cell death (Telford, 2012). Major flow
cytometric assays include membrane asymmetry (e.g., annexin V and 2.1.1 Reagent preparation
F2N12S staining assays), membrane permeability (e.g., nucleic acid and
inclusion and exclusion dyes), and mitochondria assays. To perform the trypan blue stain assay, 0.4% trypan blue stain and
When selecting the appropriate cell viability assay, factors including phosphate-buffered saline (PBS) or serum-free medium are obtained.
cost, speed, sensitivity, and the required equipment should be consid- Trypan blue stain should be stored in dark and filtered after prolonged
ered in order to obtain reliable results (Shokrzadeh & Modanloo, 2017). storage. As trypan blue stain binds to serum proteins and causing mis-
An ideal cell viability assay should be safe, rapid, reliable, efficient, and leading results, serum-free medium should be used to obtain reliable
time- and cost-effective, and should not interfere with the test com- results.
pound (Aslantürk, 2018). On the other hand, regardless of the assay
chosen, the most critical factors for accurate and reproducible mea-
surements include (a) the use of a controlled and consistent source of 2.1.2 Protocol
cells to set up experiments and (b) performing suitable characterization
of reagent concentration and incubation time for each experimental The cell suspension to be tested is centrifuged at 100 × g for 5 min.
model system (Riss et al., 2016). Considering the above, in this guide- The supernatant is discarded and the pellet is resuspended in 1-ml
334 KAMILOGLU ET AL .
PBS solution or serum-free medium. Then, one portion of this cell sus- stained red and unstained, respectively, are counted under light micro-
pension is mixed with one portion of trypan blue stain. The mixture is scope.
allowed to stay at room temperature for 3 min. It is important to note
that the cells should be counted within 3–5 min of mixing with try-
pan blue, as longer incubation periods will lead to cell death and hence 2.2.3 Calculation
reduced viability counts. Following the incubation, a drop of the mix-
ture is applied to a hemocytometer, which is placed on the stage of a The calculations are carried out as in trypan blue stain assay using the
binocular microscope. Viable, that is, unstained, and nonviable, that is, following equation:
stained, cells in the hemocytometer are counted separately.
% Viable cells
Total number of viable cells per milliliter of aliquot
= × 100.
2.1.3 Calculation Total number of cells per milliliter of aliquot
After counting viable and nonviable cells, the total number of viable
cells per milliliter of aliquot is determined by multiplying the total num- 3 COLORIMETRIC ASSAYS
ber of viable cells by 2, which is the dilution factor for trypan blue. Sim-
ilarly, total number of cells per milliliter of aliquot is determined by 3.1 MTT assay
addition of number of viable and nonviable cells and multiplying it by
2. Then, the percentage of viable cells is calculated using the following MTT assay is a simple colorimetric test of cell proliferation and survival,
equation: which was developed by Mosmann (1983) and adapted by Cole (1986)
for measuring chemosensitivity of human lung cancer cell lines. The
% Viable cells assay is based on the conversion of MTT into formazan crystals by liv-
Total number of viable cells per milliliter of aliquot ing cells, which shows mitochondrial function (Van Meerloo, Kaspers,
= × 100. & Cloos, 2011). It is well known as the first homogeneous cell viabil-
Total number of cells per milliliter of aliquot
ity assay was designed for 96-well plates for high-throughput screen-
ing. Since then, MTT tetrazolium assay technology has been widely
2.2 Eosin, congo red and erythrosine B stain adopted and remains popular in academic labs as evidenced by thou-
assays sands of published articles (Abbott, 2003). In MTT assay, the tetra-
zolium salt is reduced to insoluble formazan dye by dehydrogenase
Eosin is a fluorescent red dye that is used to stain cytoplasm, colla- enzyme present in the viable cells at 37◦ C (Figure 2). Further, the insol-
gen, and muscle fiber, facilitating their visualization under a microscope uble formazan salt is dissolved by the addition of solubilizing agents,
(Nakayama & Tsujinaka, 2014). Similarly, congo red is a sulfonated azo and the colored product is quantitatively measured at 570 nm using a
dye that is used in microscopy to stain cytoplasm. Erythrosine B, also spectroscopic multiplate reader. A variety of methods have been used
known as FD&C Red No. 3, is a tetraiodofluorescein dye, which is to solubilize the formazan product, stabilize the color, avoid evapora-
widely utilized as biological stain and color additive in food and drugs tion, and reduce interference by phenol red and other culture medium
(Kuo et al., 2017). The principle of eosin, congo red, and erythrosine B components (Hall et al., 2004; Wilson & Hay, 2011). Various solubiliza-
stain assays also relies on the integrity of the cell membrane as in try- tion methods include the use of acidified isopropanol, dimethyl sulfox-
pan blue stain assay. Particularly, erythrosine B stain has several advan- ide (DMSO), dimethylformamide (DMF), sodium dodecyl sulfate (SDS),
tages over trypan blue stain including (a) being nontoxic, (b) not binding and combinations of detergent and organic solvent (Abbott, 2003; Hall
to serum proteins, and (c) not requiring an incubation period prior to et al., 2004; Wilson & Hay, 2011). The dead cells lose the ability to
counting (Kim et al., 2016). reduce tetrazolium salts into colored formazan products. Viable cells
with active metabolism convert MTT into a purple-colored formazan
product with an absorbance maximum near 570 nm. Thus, the intensity
2.2.1 Reagent preparation of the colored product is directly proportional to the number of viable
cells present in the culture (Präbst, Engelhardt, Ringgeler, & Hübner,
To perform these assays, eosin, congo red, or erythrosine B stain (0.1%) 2017).
and PBS are obtained from the manufacturer.
3.1.1 Reagent preparation

2.2.2 Protocol
MTT solution is prepared by dissolving MTT in Dulbecco’s phosphate-
The cell suspension in PBS and the stain are mixed at 1:1 ratio and then buffered saline (DPBS) at pH 7.4 (5 mg/ml). This solution is filtered
the mixture is loaded into a hemocytometer. Nonviable and viable cells, and sterilized through a 0.2-µm filter into a sterile and light-protected
FIGURE 2 Reduction of MTT to formazan crystals
container. MTT solution should be stored at –20◦ C until analysis or growth (Cory, Owen, Barltrop, & Cory, 1991). It is categorized in a more
at 4◦ C for immediate use and should be protected from the light. recently developed group of tetrazolium reagents that can be reduced
Solubilization solution is prepared with 40% (v/v) DMF containing 2% by viable cells to produce formazan products directly soluble in the
(v/v) glacial acetic acid under ventilated fume hood. SDS (16% [w/v]) is cell culture medium (Barltrop, Owen, Cory, & Cory, 1991; Goodwin,
added to this solution and pH is adjusted to 4.7. Solubilization solution Holt, Downes, & Marshall, 1995). Such enhanced tetrazolium reagent
should be stored at room temperature in order to prevent precipita- removes liquid handling during the assay process because a second
tion of SDS and in case of precipitation it can be heated to 37◦ C for application of the reagent to the assay plate is not required to solubilize
resolubilization. the precipitate of formazan, rendering the protocols easier. This group
of tetrazolium reagents is also used in combined application with inter-
mediate electron-acceptor reagents such as PMS (phenazine methyl
3.1.2 Protocol sulfate) or PES (phenazine ethyl sulfate) that can penetrate viable cells,
lessen cytoplasm, or cell surface, and exit the cells in which tetrazolium
Cell suspensions seeded to 96-well plates (100 µl/well) with or without can be converted to a dissolved formazan product (Berridge, Herst, &
the test compounds are incubated at 37◦ C in a humidified incubator Tan, 2005).
with 5% CO2 for required exposure time. MTT solution of 10 µl is
added to each well to reach a final concentration of 0.45 mg/ml and
incubated at 37◦ C for 1–4 hr. After incubation, the formazan crystals 3.2.1 Reagent preparation
are dissolved in 100 µl of solubilization solution and the absorbance is
measured at 570 nm with a multiplate reader. MTS solution is prepared by dissolving 2 mg/ml of MTS powder in
DPBS until a clear yellow solution is obtained. PES powder is dissolved
in MTS solution to 0.21 mg/ml, and the pH is adjusted to 6.0–6.5 with
3.1.3 Calculation 1 N HCl. This solution is filtered and sterilized through a 0.2 µm filter
into a sterile and light-protected container. MTS solution should be
The percentage of cell viability is calculated using the following equa- stored at –20◦ C until analysis or at 4◦ C for immediate use and should
tion: be protected from light.
% Viability
Mean ODsample 3.2.2 Protocol

= × 100.
Mean ODblank
Cell suspensions seeded to 96-well plates (100 µl/well) with or without
the test compounds are incubated at 37◦ C in a humidified incubator
3.2 MTS assay with 5% CO2 for required exposure time. MTS solution of 20 µl is
added to each well to reach a final concentration of 0.33 mg/ml and
MTS has initially been developed as a new tetrazolium analog of MTT as incubated at 37◦ C for 1–4 hr. After incubation, the absorbance is
a substitute for MTT in the microculture screening assay for in vitro cell measured at 490 nm with a multiplate reader.
3.2.3 Calculation 3.4 WST-1 assay
The percentage of cell viability is calculated using the following equa- WST-1 has been developed by Ishiyama, Shiga, Sasamoto, Mizoguchi,
tion: and He (1993). It is a tetrazolium salt that produces a highly water-
soluble formazan by mitochondrial dehydrogenase enzymes in the
Mean ODsample
% Viability = × 100. presence of intermediate electron acceptor, such as 1-methoxy PMS.
Mean ODblank
The amount of formazan produced is directly proportional to the
amount of mitochondrial dehydrogenase in cell culture. Thus, the assay
3.3 XTT assay measures the metabolic activity of cells. WST-1 has a similar sensitivity
to XTT, whereas it is less toxic compared to XTT. Furthermore, WST-
XTT has been synthesized by Paull et al. (1988). Bioreduction of XTT 1 does not require an additional step to dissolve the formazan as in
yields a highly colored formazan product with only viable cells. In con- case of MTT, which is advantageous when large-scale drug screening
trast to other tetrazolium salts such as MTT, formazan dye is soluble in is attempted (Ishiyama et al., 1993).
aqueous solutions and directly quantified using a scanning multiplate

spectrophotometer (ELISA reader). This enables a high degree of accu-
racy, allows online data processing by computers (data collection, cal- 3.4.1 Reagent preparation
culation, and reporting generation), and thus allows a high number of
samples to be handled quickly and conveniently. Cells are incubated WST-1 reagent solution is prepared as an aqueous solution con-
with the yellow XTT solution in a 96-well tissue culture plate. Dur- taining 5 mM WST-1, 0.2 mM 1-methoxy PMS, and 12.5 mM 4-(2-
ing this time of incubation, orange formazan solution is produced and hydroxyethyl)-1-piperazineethanesulfonic acid buffer (pH 7.0).
is quantified spectrophotometrically utilizing an ELISA plate test. An

increase in the number of living cells results in an increase of the sam-
ple’s total activity of mitochondrial dehydrogenases. This rise is closely 3.4.2 Protocol
associated with the quantity of formed orange formazan, as measured
by the absorbance (Paull et al., 1988; Scudiero et al., 1988). Cell suspensions seeded to 96-well plates (100 µl/well) with or without
with 5% CO2 for required exposure time. Then, 10 µl of WST-1 reagent
solution is added to each well and the plate is incubated at 37◦ C for
2 hr. After incubation, the absorbance is measured at 450 nm with a
multiplate reader.
XTT working solution was prepared, immediately before use, by dis-
solving l mg/ml XTT in sterile Hanks’ balanced salt solution followed by
addition of PMS at 5 µL/ml (5 mM stock solution).
3.4.3 Calculation
The percentage of cell viability is calculated using the following equa-

3.3.2 Protocol
tion:
Cell suspensions seeded to 96-well plates (10,000 cells/well) with or Mean ODsample
% Viability = × 100.
without the test compounds (200 µl/well) are incubated at 37◦ C in a Mean ODblank
humidified incubator with 5% CO2 for required exposure time. At the
end of incubation, 100 µl of XTT solution mix was added to each well
(final concentration = 0.3 mg/ml), and plates were incubated at 37◦ C
3.5 WST-8 assay
for 4 hr. Absorbance was measured at 450 nm against a reference
WST-8 is a second-generation tetrazolium salt that was first synthe-
wavelength at 650 nm using a microplate reader.
sized by Tominaga et al. (1999). It is used as a chromogenic indicator
for cell viability. The reduction of slightly yellow WST-8 by viable
3.3.3 Calculation cells produces an orange-colored formazan product, which is directly

proportional to the number of viable cells in the range of 200–25,000
The percentage of cell viability is calculated using the following equa- cells/well for many cell lines including nonadherent cells. WST-8
tion: is found to be more sensitive for cell viability measurements than

those of other tetrazolium salts including MTT, MTS, XTT, and WST-1.
(A450 − A650 ) of test cells Furthermore, WST-8 produces water-soluble formazan upon cellular
(A450 − A650 ) of control cells reduction, which does not require an additional step to dissolve the
To preform LDH assay, substrate, lysis, and stop solutions are prepared.
For the preparation of substrate solution, first INT and 1-methoxy PMS
(1-methoxyphenazine methosulfate) are dissolved in PBS to a final
concentration of 100 mM. Then, 0.054 M L-(+)-lactic acid, 1.3 mM β-
F I G U R E 3 Conversion of lactate to pyruvate catalyzed by lactate
NAD+, 0.66 mM INT, and 0.28 M 1-methoxy PMS solutions dissolved in
dehydrogenase (LDH)
0.2 M Tris–HCl buffer at pH 8.2 are combined to obtain the substrate
formazan, providing an additional advantage to the method (Tominaga solution. It is important to note that the substrate solution should be
et al., 1999). prepared as fresh prior to each experiment. The lysis solution is pre-
pared with 9% (v/v) Triton X-100, whereas to prepare the stop solu-
tion, 50% DMF and 20% SDS at pH 4.7 are used. Alternatively, 1 N
3.5.1 Reagent preparation HCl can also be used to stop the reaction; however, DMF–SDS solu-
tion is advantageous in cell culture medium containing Phenol Red, as
WST-8 reagent solution is prepared as an aqueous solution containing it neutralizes the background absorption. Interference of background
5 mM WST-8, 0.2 mM 1-methoxy PMS, and 150 mM NaCl. absorption can also be eliminated using a Phenol Red- free medium.
3.6.2 Protocol
3.5.2 Protocol
As different cell lines comprise diverse amounts of LDH, a preliminary
Cell suspensions seeded to 96-well plates (100 µl/well) with or without
study should be performed to ensure the optimum number of cells.
Accordingly, various cell dilutions (0–20,000 cells/well) are prepared
with 5% CO2 for required exposure time. Then, 10 µL of WST-8
and 100 µl of the cell suspensions are added to 96-well plate. After-
reagent solution is added to each well and the plate is incubated at
ward, 15 µl of lysis solution is added to each well and the plate is cen-
37◦ C for 2 hr. After incubation, the absorbance is measured at 450 nm
trifuged at 250 × g for 4 min. Then, 50 µl of the supernatants are trans-
with a multiplate reader.
ferred to 96-well flat-bottom enzymatic assay plate followed by the
addition of 50 µl substrate solution. The plate is covered to protect it
against the light and incubated at 37◦ C for 15–30 min. After incuba-
3.5.3 Calculation
tion, 100 µl of stop solution is added and within 1 hr of stop solution
addition, the absorbance is measured at 490 nm using a plate reader.
The percentage of cell viability is calculated using the following equa-
The background absorbance is set at 690 nm and subtracted from the
tion:
measurements carried out at 490 nm. At this point, it is important to
Mean ODsample ensure that no bubbles are formed in the wells. The cell concentration
Mean ODblank with absorbance values at least two times the background absorbance
of the control medium is determined as the optimum cell number. The
assay should be performed at least in triplicates.
3.6 LDH assay
LDH assay was developed in the 1980s as a rapid and sensitive method 3.6.3 Calculation
for assaying cytotoxicity in immune cells (Decker & Lohmann-Matthes,
1988). LDH is a stable cytoplasmic enzyme that is released into the The absorbance values obtained for the optimum cell concentration is
cell culture medium due to the loss of membrane integrity (Chan, used for the determination of the percent of cell death (% cytotoxicity)
Moriwaki, & De Rosa, 2013), which is a typical characteristic of cells using the following equation:
undergoing apoptosis, necrosis, or other forms of cellular damage. LDH
Experimental LDH release (OD490 )
oxidizes reduced form of nicotinamide adenine dinucleotide (NADH), % Cytotoxicity = × 100.
Maximum LDH release (OD490 )
generating NAD+, and catalyzes the conversion of lactate to pyruvate
(Figure 3). In this protocol, NADH reduces the yellow tetrazolium salt, Alternatively, LDH standard can be used to express the results.
INT (iodonitrotetrazolium or 2-(4-iodophenyl)-3-(4-nitrophenyl)-5- Accordingly, 50 µl of assay substrate is added to 50 µl of different dilu-
phenyl-2H-tetrazolium), into a water-soluble red formazan dye. The tions of LDH standard dissolved in cell culture medium (0.2–2.0 U/ml).
amount of formazan is determined considering the absorbance values The mixture is incubated for 15 min and the absorbance values are
measured at 490 nm, which represents the total LDH activity in the measured as indicated above. A standard curve is plotted using the
culture and it is directly proportional to the number of damaged cells obtained data and it is used to calculate the enzyme activity of the
(Kumar, Nagarajan, & Uchil, 2018). tested samples.
3.7 SRB assay 3.8 NRU assay
SRB assay has initially been developed in 1990 to evaluate the cyto- NRU assay has been developed to quantify viable cells in monolayer
toxicity of anticancer drugs (Skehan et al., 1990). SRB is a bright-pink cultures (Borenfreund & Puerner, 1985). This protocol is based on
aminoxanthene dye with two sulfonic groups that bind to amino-acid the binding ability of viable cells to the supravital dye neutral red
residues under mild acidic conditions, and dissociate under basic condi- (3-amino-7-dimethylamino-2-methyl-phenazine hydrochloride) in the
tions. This protocol is based on the binding ability of SRB to cellular pro- lysosomes. The bound dye is then extracted from the viable cells using
teins, which is fixed using TCA (trichloroacetic acid). The protein-bound an acidified ethanol solution, and the absorbance of the solubilized dye
dye is then dissolved in Tris-base (tris(hydroxymethyl)aminomethane) is measured using a spectrophotometer (Repetto, Del Peso, & Zurita,
solution and the absorbance values measured at 510 nm are 2008).
used to determine the number of viable cells (Vichai & Kirtikara,
2006).
3.7.1 Reagent preparation To preform NRU assay, neutral red working solution dissolved in PBS
(40 µg/ml) and destain solutions are prepared. Neutral red destain solu-
To preform SRB assay, 10% (w/v) TCA solution, 0.057% (w/v) SRB dis- tion contained 50% ethanol (96%), 49% deionized water, and 1% glacial
solved in 1% (v/v) acetic acid, 1% (v/v) acetic acid solution, and 10 mM acetic acid (v/v). In addition, optionally, 5% glutaraldehyde may be pre-
unbuffered Tris-base solution at pH 10.5 are prepared for fixation, pared by dilution of the commercial 25% for fixation purpose.
staining, washing, and dissolution steps, respectively.
3.8.2 Protocol
3.7.2 Protocol
Cell suspensions seeded to 96-well plates (approximately 50,000
Cell suspensions seeded to 96-well plates (19,000 cells/well) with or cells/well) are incubated overnight to form a half-confluent monolayer.
without the test compound (10 µl dissolved in 10% DMSO) are incu- Next day, the medium is removed from the plate and the cells are
bated at 37◦ C in a humidified incubator with 5% CO2 for 72 hr. The treated or untreated with the test compounds, followed by another
cells attached to the bottom of the wells are fixed with addition of 100 incubation for overnight. At this stage, neutral red working solution is
µl of cold TCA and subsequent incubation at 4◦ C for 1 hr. After 1 hr, also prepared and incubated overnight at the same temperature as the
the plate is washed four times with slow-running tap water. The excess cells. Following incubation, the neutral red solution is centrifuged at
water is removed using paper towel and a blow dryer is used to dry the 600 × g for 10 min to remove any precipitated dye crystals. The medium
plate completely. Afterward, 100 µl of the SRB solution is added to the is removed from the cells and 100 µL of neutral red solution is added
cells and the plate is incubated at room temperature for 30 min. After to each well. The plate is incubated for 2 hr under proper culture con-
30 min of incubation, the unbound SRB stain is removed by washing the ditions and then the red medium is removed and the cells are washed
wells four times with acetic acid solution. Again, a blow dryer is used to with 150 µl PBS. For low-adherent cell cultures, it is recommended to
completely dry the plate and then SRB stained cells are dissolved in 200 also include a fixation step with 5% glutaraldehyde for 2 min prior to
µl of unbuffered Tris-base solution. The plate is shaken for 5 min to sol- washing. Afterward, 150 µl of neutral red detain solution is added to
ubilize the protein-bound SRB dye. Another option is to incubate the each well and the plate is shaken for 10 min to extract the neutral red
plate for 30 min in Tris-base solution for complete solubilization of SRB dye from the cells and to obtain a homogeneous solution. Then, the
dye. Then, the absorbance values are recorded at 510 nm using a plate absorbance values are recorded at 540 nm with a plate reader spec-
reader. Alternatively, the measurement can be done fluorometrically at trophotometer using blanks that contain no cells as a reference. Alter-
excitation and emission wavelengths of 488 and 585 nm, respectively. natively, the measurement can be done fluorometrically at excitation
The assay should be performed at least in triplicates. and emission wavelengths of 530 and 645 nm, respectively. The assay
should be performed at least in triplicates.
3.7.3 Calculation
3.8.3 Calculation
The percentage of cell-growth and growth inhibition are calculated
using the following equations: The percentage of cell viability is calculated using the following equa-
tion:
Mean ODsample
% Cell growth = × 100,
Mean ODblank Mean ODsample
% Growth inhibition = 100 − % Cell growth. Mean ODblank
3.9 CVS assay 4 FLUOROMETRIC ASSAYS
CVS assay was developed by Saotome, Morita, and Umeda (1989) Fluorometric assays are developed in 1990s as an alternative to exclu-
for evaluating the cytotoxicity of chemicals. It is used for the indirect sion dyes and colorimetric methods. Fluorometric cell viability meth-
quantification of cell death. The protocol is based on the determination ods are based on the nonspecific cleavage of a nonfluorescent com-
of the adherence of cells through staining of attached cells with CVS, pound such as fluorescein diacetate which fluoresces following its
which binds to proteins and DNA. The dead cells lose their ability cleavage by cellular esterases. Nascent fluorescent signal is then mea-
to adhere, which results in the reduction of the amount of CVS in sured to determine the amount or the ratio of the viable cells. Fluoro-
the cell culture. The bound dye is extracted from the viable cells metric assays are easy to perform and relatively cheap but fluorescent
using methanol, and the absorbance of the solubilized dye is mea- interference caused by the applied test compounds is possible (Altman,
sured using a spectrophotometer (Feoktistova, Geserick, & Leverkus, Randers, & Rao, 1993; Rotman & Papermaster, 1966).
2016).
4.1 Resazurin (alamar blue) assay

The resazurin-based test was first used to examine the sanitary state
To preform CVS assay, 0.5% CVS solution is prepared by dissolving of milk in the late 1920s (Ali-Vehmas, Louhi, & Sandholm, 1991;
crystal violet powder in 20% aqueous methanol. CVS solution can be Nixon & Lamb, 1945; Twigg, 1945). After that, it has been used for
stored at room temperature up to 2 months. plant metabolism studies (De Jong & Woodlief, 1977), semen quality
evaluations (Glass et al., 1991), and antifungal susceptibility tests (To,
Fothergill, & Rinaldi, 1995). It has also been a valuable method for
3.9.2 Protocol analyzing toxicants owing to several advantages of the assay (Hamid,
Rotshteyn, Rabadi, Parikh, & Bullock, 2004; O’Brien, Wilson, Orton, &
Cell suspensions seeded to 96-well plates (10,000–20,000 cells/well) Pognan, 2000; Perrot, Dutertre-Catella, Martin, Rat, & Warnet, 2003).
are incubated at 37◦ C for 18–24 hr to enable adhesion of cells to wells. Moreover, the safety, simplicity, homogeneity, and sensitivity of this
Wells without cells are also prepared to serve as a control to avoid non- method offer predominance over the other classic tests used to mea-
specific binding of the CVS. After incubation, the medium is removed sure cell viability and proliferation (Jonsson, Frost, Larsson, Ljunghall,
from the plate and 100 µl of test compound at varying concentrations & Ljunggren, 1997; Larson, Doughman, Gregerson, & Obritsch, 1997).
are added to the wells, followed by another incubation at 37◦ C for 18– Alamar blue fluorometric assay is based on the nonspecific, enzy-
24 hr. Afterward, the medium is removed and the plate is washed two matic, irreversible reduction of the compound by viable cells. Follow-
times with slow-running tap water. The excess water is removed gen- ing the enzymatic reaction within the cells, alamar blue or resazurin is
tly by inverting the plate on a filter paper. Then, 50 µl of CVS solu- reduced into resorufin, which is pink and extracted from the living cells
tion is added to each well and the plate is incubated at room temper- into the medium. The extracted compound results in a change in the
ature for 20 min on a bench rocker with a frequency of 20 oscillations color of the medium and color change can be measured from 50 up to
per minute. After 20 min of incubation, the unbound CVS is removed 50,000 cells in a linear range, using 530–570 nm for excitation/580–
by washing the wells four times with tap water. Again, the plate is 620 nm for emission fluorescent filters. The alamar blue method is
inverted on a filter paper to remove excess water and then air-dried sensitive, simple, and safe to monitor the cell viability and prolifera-
for minimum of 2 hr. Once the plate becomes completely dry, 200 µl tion (Czekanska, 2011; Johnson, Nguyen, & Coder, 2013; Larson et al.,
of methanol is added to each well and incubated at room temperature 1997). There are several commercially available alamar blue cell viabil-
for 20 min on a bench rocker with a frequency of 20 oscillations per ity and proliferation kits such as alamarBlue Cell Viability Reagent from
minute. Then, the absorbance values are recorded at 570 nm using a Thermo Fisher Scientific, alamarBlue from Bio-Rad, in vitro Toxicology
plate reader. The assay should be performed at least in triplicates. Assay Kit from Sigma–Aldrich, AlamarBlue Cell Viability Assay Reagent
from G-Biosciences, Cell-Quant AlamarBlue Cell Viability Reagent
from GeneCopoeia, and so forth.
3.9.3 Calculation
The average absorbance values of wells without cells are subtracted 4.1.1 Reagent preparation
from the absorbance values of wells with cells. Then, the percentage
of cell viability is calculated using the following equation: Alamar blue solution can also be prepared from the powder instead of
using commercially available kits. Alamar Blue high-purity powder is
Mean ODsample dissolved in PBS (pH 7.4) to 0.15 mg/ml. Alamar blue solution can be
Mean ODblank sterilized via filtering and can be stored at 4◦ C for short-term storage
and at –20◦ C for long-term storage (Riss et al., 2016).
F I G U R E 4 Schematic illustration of the principles of 5-carboxyfluorescein diacetate acetoxymethyl ester (5-CFDA-AM) assay. 5-CFDA-AM is
a target of intracellular nonspecific esterase enzymes in living cells. Following the nonspecific enzymatic activity of esterases, 5-CFDA-AM is
converted into fluorescent substance carboxyfluorescein (CF), which is polar and nonpermeable through the cellular membrane of living cells.
Dead or dying cells lack the esterases and membrane permeability control so that they do not have any fluorescent signal
4.1.2 Protocol sured at excitation and emission wavelengths of 530–570 and 580–
620 nm, respectively, on the plate reader. For calculation, average value
Alamar blue cell viability and proliferation assay can be applied to a of no-cell control is subtracted from each well. A cell number standard
number of cell types but it is not recommended to run the assay imme- curve should be prepared for each cell type for the analysis of obtained
diately after thawing the cells from cryopreservation. Cell density opti- values if the exact cell number calculation is necessary (Czekanska,
mizations should be assessed before running the assay depending on 2011; Riss et al., 2016). In addition to exact cell number calculation, the
the cell type (adherent/suspension) and cell size (hepatocytes, stem percentage of cell growth and growth inhibition can also be calculated
cells or neuronal cells, etc.). using the equations described in the previous sections.
After splitting the cells into the corresponding wells with the culture
medium, cells are incubated at least for 5 hr at 37◦ C, supplemented
with 5% CO2 . Incubation time can be adjusted for treatment conditions 4.2 5-CFDA-AM assay
and can be longer. Noncell controls are included to check the back-
ground fluorescence and nontreated/sham-treated controls constitute 5-CFDA-AM is another compound used in fluorometric cell viability
the negative controls. Following the incubation, 10% (v/v) alamar blue assays. Similar to alamar blue, 5-CFDA-AM is a target of intracellular
solution is added into the each well. If the cells are adherent, culture nonspecific esterase enzymes in living cells. Following the nonspecific
medium can be aspirated and replaced by a new medium mixture con- enzymatic activity of esterases, 5-CFDA-AM is converted into fluores-
taining 10% (v/v) alamar blue. Alamar blue solution should be kept in a cent substance carboxyfluorescein, which is polar and nonpermeable
light-shield tube and once the solution is added into the wells, culture through the cellular membrane of living cells (Ganassin & Bols, 2000)
plates should be covered with aluminum folio. Cells are incubated with (Figure 4). Examples of commercially available 5-CFDA-AM probes
alamar blue solution for 4 hr at 37◦ C, supplemented with 5% CO2 . are as follows: 5-CFDA-AM (5 mg) from Invitrogen, 5-CFDA, AM from
After the incubation, 150 µl medium is transferred into a new Synchem, 5-CFDA (5-Carboxyfluorescein diacetate, single isomer,
96-well plate (duplicates for each sample) and fluorescence is mea- 100 mg) from Biotium, and so forth.
4.2.1 Reagent preparation ber and measured luminescence using the luciferin–luciferase reaction
(Crouch, Kozlowski, Slater, & Fletcher, 1993). Intracellular ATP is a valid
5-CFDA, AM stock solution of 4 mM can be prepared in anhydrous indicator of cell viability. ATP synthesis is interrupted and remaining
DMSO. Stock solution can be aliquoted and stored at –20◦ C but the ATP is depleted by ATPases immediately once the cells loose mem-
solution must be protected from light and moisture (Ganassin & Bols, brane integrity and cell viability (Ugarova, Brovko, Trdatian, & Raı̆nina,
2000). 1987). Intracellular ATP concentration may vary with the cellular stress
factors, physiological changes such as treatments, and cell viability
(Ugarova, 1993). For the detection of ATP concentration, luciferase
4.2.2 Protocol assay can be applied as ATP is a necessary component for the oxida-
tion reaction of luciferin (McElroy, 1947). ATP-coupled luciferase reac-
Cells should be plated into culture plates that are also compatible tion can be summarized as follows (Lomakina, Modestova, & Ugarova,
with plate readers. Wells without cells should be included to monitor 2015):
the background fluorescence. Stock solution of 4 mM 5-CFDA, AM is
Firefly luciferase
diluted (1:1,000) using the serum- and amino acid-free culture medium. D−Luciferin + O2 + ATP + Mg+2 → oxyluciferin
Culture medium is aspirated from the wells of adherent cells and
+ AMP + PPi + lightquantum (∼ 560nm) .
diluted 5-CFDA, AM solution, 4 µM working solution, is added to
the respective cells. For suspension cell cultures, 8 µM 5-CFDA, AM
In the luminometric ATP cell viability assays, cells first get perme-
working solution can be prepared via 1:500 dilution of 4 mM stock
able to ATP so that luciferase enzyme can interact with intracellular
solution in serum- and amino acid-free culture medium. Cells are split
ATP. Then intracellular ATPases are inactivated and finally the light
into the wells using serum- and amino acid-free culture medium and
is measured via luminometers to determine the intracellular ATP
then equal amount of 8 µM 5-CFDA, AM working solution is added to
levels (Figure 5). Luminescent signal is quite stable and can be mea-
the corresponding wells. For example, for 96-well plate cultures, cells
sured within a few hours and most of the assays are very specific
are split into wells within 100 µl serum- and amino acid-free culture
that the signal can be measured even from 50 cells. Examples of
medium and then 100 µl of 8 µM 5-CFDA, AM working solution is
commercially available luminometric ATP assays are as follows: ATP
added to each well.
Assay Kit – Luminometric from Assay Biotech, ATP Determination Kit
Cells are incubated with 5-CFDA, AM for 30 min at 18–22◦ C in the
from ThermoScientific, Luminescent ATP Detection Assay Kit from
dark. Following incubation, fluorescence is measured at excitation and
Abcam, CellTiter-Glo Luminescent Cell Viability Assay from Promega,
emission wavelengths of 493 and 541 nm, respectively, using a fluores-
Rapid Luminometric ATP Assay Kit from AAT Bioquest, and so
cence plate reader (Ganassin & Bols, 2000; Schirmer, Chan, Greenberg,
forth.
Dixon, & Bols, 1997; Schreer, Tinson, Sherry, & Schirmer, 2005).
The percentage of cell growth and growth inhibition can be calcu-
lated using the equations described in the previous sections. If exact
cell number calculation is necessary, a cell number standard curve
5.1.1 Protocol
should be prepared for each cell type for the analysis of obtained
Commercially available luminometric ATP cell viability assays are rel-
values.
atively cheap, easy to perform, minimize the technical errors, and give
reproducible data, so that the manual preparation of the assay is almost
5 LUMINOMETRIC ASSAYS fully replaced by the kits. In general, kits are designed for 96- or 384-
well plates.
The bioluminescence assays are based on the correlation between Cells are split into 96-well plates in 100 µl (25 µl for 384-well plate)
a bioluminescent reaction and the effect of a tested compound. This culture medium containing desired treatment compounds or condi-
effect can be an increase in cell proliferation or cell death (Bulich tions. Like previously described cell viability assays, no-cell wells should
& Isenberg, 1981; Voyevodina, Nifantyev, Kovalevsky, Schultz, & be included for blank subtraction and no-treatment cells should be
Kratasyuk, 1990). Bioluminescent measurements are performed using included as controls. After desired incubation or treatment period,
luminometers since 1970s. Modern luminometers carry a photon plates are equilibrated to room temperature for 30 min. Most of
counter and the obtained signal is proportional, but not equal, to the the ready-to-use kits have only one working solution containing all
emitted photons (Lundin, 2000). compounds of the assay: detergents, ATPase inhibitors, luciferin, and
luciferase (Assay Biotech, AAT Bioquest, Promega, and ThermoSci-
entific [Abcam, Cambridge, UK]). After incubation at room tempera-
5.1 ATP assay ture, equal volume of assay buffer (100 µl for 96-well plate and 25 µl
for 384-well plate) is added to each well and incubated on an orbital
ATP bioluminescence has initially been developed to determine shaker at room temperature for 10–20 min. If the kit has separate solu-
whether there was a linear relationship between cultured cell num- tions (like Luminescent ATP Detection Assay Kit from Abcam), 50 µl
F I G U R E 5 Schematic illustration of the principles of ATP assay. ATP is an essential component of living cells. ATP synthesis is interrupted and
remaining ATP is depleted by ATPases immediately once the cells loose membrane integrity and cell viability. In the luminometric ATP cell viability
assays, cells first get permeable to ATP so that luciferase enzyme can interact with intracellular ATP. ATP is a necessary component for the
oxidation reaction of luciferin and coupled reaction results in oxyluciferin, pyrophosphate, and light. The light is measured via luminometers to
determine the intracellular ATP levels
detergent–ATPase inhibitor buffer is added first and after incubation ATP. Instead, viable cells uptake pro-substrate and convert it into “sub-
50 µl luciferin–luciferase buffer is added and incubated (96-well plate). strate” that diffuses into the culture medium. Then, luciferase enzyme
For calculation, ATP standard curve is prepared using the standard uses diffused substrate and generates luminescent signal. This method
ATP stock solution. A standard curve with 10 pM to 10 µM range is can be applied for both continuous measurement applications and end-
anticipated to be sufficient for comparison. Working solution is also point assays (Aslantürk & Çelik, 2017; Riss et al., 2016).
added to the standard curve wells and the standard curve plate is incu- Examples of commercial real-time viability assay kits are RealTime-
bated at room temperature for the same period as the experimental Glo MT Cell Viability Assay from Promega and MSCGlo Real Time from
groups. HemoGenix.
Following incubation, luminescence is measured by a luminometer
at 560 nm wavelength.
5.2.1 Protocol
5.2 Real-time viability assay Cells are split into the wells with medium containing desired treat-
ment compound and real-time viability assay buffer. Cells are then
Real-time viability assay is a new approach of luciferase method and is incubated at 37◦ C and continuous luminescence measurements are
the only cell viability method that allows to monitor the cell viability in recorded every 30 min or every hour depending on the experimental
real time. In this new approach, a marine shrimp-derived engineered design. Real-time cell viability assay buffers are stable at 37◦ C up
luciferase and a pro-substrate of luciferase are used. In this method, to 72 hr but an optimization step is necessary for each cell type to
cell-permeable pro-substrate and luciferase are added into the cul- determine the time period in which limiting factor pro-substrate is
ture medium as well but cells are not lysed to release the intracellular consumed totally. For end-point experiments, real-time viability buffer
F I G U R E 6 Surface staining of mouse splenocytes combined with LIVE/DEAD Fixable Aqua Dead Cell Stain (ThermoFisher Scientific). A,
Gating strategy may start with eliminating the dead cells first and continue with FSC–SSC and phenotyping markers CD3 and CD8. B, Gating can
also start with selecting populations with FSC–SSC and phenotyping markers CD3 and CD8. After selecting CD8+ cells, the viable population is
selected via eliminating Aqua positive cells. Splenocytes were analyzed with FACS Canto and FlowJo Software (10.6.2. for Windows 10, ©Becton,
Dickinson and Company [BD]) was used to analyze flow cytometry data. FITC antimouse-CD3 clone 17A2 and APC/Cyanine7 antimouse CD8a
clone 53-6.7 were both from Biolegend (unpublished data)
is added into the wells at the end of the treatment conditions. Cells 6.1 Membrane asymmetry assays
are incubated with the buffer at 37◦ C for 10 min to 1 hr and then
luminescent signal is measure (Riss et al., 2016). For cell viability cal- In the early stages of the cell death, especially with apoptosis, cells
culation, standard ATP measurement or percentage cell growth can be preserve the membrane permeability and integrity, but the composi-
used. tion of the outer surface of cell membrane is altered: phospholipids
become loosely packed and phosphatidylserine starts to appear at the
outer surface of the cell membrane. Outer surface phosphatidylserines
6 FLOW CYTOMETRIC ASSAYS can be detected by fluorescently labeled annexin V, which is a Ca2+ -
dependent phospholipid-binding protein (Van Engeland, Ramaekers,
Development of first flow cytometry machines dates back to 1950s. Schutte, & Reutelingsperger, 1996).
First flow cytometry was designed as simple cell counter but two- A more sophisticated solution for the detection of the changes on
parameter flow cytometry applications were developed immediately. cellular membrane can be obtained via wavelength ratiometry (Dem-
The principle of flow cytometry is characterizing or phenotyping the chenko, 2010). F2N12S (4′-N,N-diethylamino-6-(N-dodecyl-N-methyl-
cells within a liquid flow through lasers. In other words, flow cytom- N-(3-sulfopropyl))ammoniomethyl-3-hydroxyflavone) probe is a small
etry is a quantitative single cell analysis. Cells can be characterized molecule that has dual-color fluorescence emission. As a result of its
depending on the size, granularity, and whether carrying a specific flu- dual-color fluorescence emission, F2N12S is a self-referencing probe;
orescent molecule or not (Macey, 1988). Flow cytometry method has obtained ratio is independent of cell size or probe concentration.
been extensively applied to cell viability and toxicity studies. First of F2N12S probe is incorporated into the cell membrane phospholipids
all, changes in forward versus side scatter (FSc vs. SSc) can be analyzed and produce green emission (at 515–545 nm). Once the cells become
without using any fluorescent dyes; dying cells are usually smaller than apoptotic, cellular membrane charges are altered and green emission
viable cells and they present increased SSc. Fluorescent staining tar- shifts toward orange emission (at 564–606 nm). The ratio of green to
geting different properties of living and healthy cells is performed for orange emission is used to measure viability percentage or ratio. As
more specific phenotypic cell viability analysis. In flow cytometry, cell F2N12S probe is a small molecule, it is not sensitive to proteases. The
viability and toxicity analysis can be easily combined with other phe- staining takes only 5 min and F2N12S binding is Ca2+ independent
notypic stainings to determine the differentiation status of a cell type, (Demchenko, 2013).
to analyze a specific cell type from a mixed culture, to determine the Both annexin V and F2N12S probe target cell surface so that
immune activation status, and so forth (Figure 6). membrane permeabilization is not required and both stainings can be
combined with other markers either for cell surface phenotyping or fixation, cells are washed again and can be used for further permeabi-
for intracellular functional analysis. lization or phenotyping steps.
Both annexin V kits can be found from different distributers.
A few examples of annexin V kits are as follows: Annexin V Stain-
ing Kits from Abcam (APC, FITC), TACS Annexin V-FITC Apop- 6.2 Membrane permeability assays
tosis Detection Kit from Biotechne, Annexin V kit from Bio-Rad
(FITC, Biotin, PE, APC), Alexa Fluor 488 Annexin V/Dead Cell Dying cells loose membrane integrity, whereas living cells are
Apoptosis Kit from ThermoFisher Scientific, Annexin V-FITC Apop- very strict with their membrane permeability. Analyzing membrane
tosis Detection Kit from Sigma–Aldrich, and so forth. F2N12S integrity is of great importance not only for mammalian cell culture
probe kit, Violet Ratiometric Membrane Asymmetry Probe/Dead studies to decide cell viability but also for food production research
Cell Apoptosis Kit for flow cytometry, is from ThermoFisher such as analyzing ethanol stress during wine production, for fertility
Scientific. labs to monitor the membrane integrity of sperms, and so forth (Da
Silveira, San Romao, Loureiro-Dias, Rombouts, & Abee, 2002; Nagy,
Hallap, Johannisson, & Rodriguez-Martinez, 2004). It is very crucial to
6.1.1 Protocol remember that apoptotic cells keep their membrane integrity and they
are resistant to exclusion dyes. On the other hand, necrotic cells can be
Annexin V and F2N12S staining protocols are quite similar to each detected with exclusion dyes (Darzynkiewicz et al., 1992). Membrane
other like all other flow cytometry surface staining protocols but there permeability assays are usually a combination of two types of dyes: one
are two important considerations: (a) annexin V binding is Ca2+ depen- staining the dead cells and a second one staining living cells.
dent and buffers should not contain any calcium chelating agents such Membrane permeability dyes can be categorized as exclusion dyes,
as EDTA; (b) F2N12S staining is sensitive to buffers containing protein inclusion dyes, and monomeric cyanine nucleic acid stains.
such as BSA or FBS.
Cells are washed with PBS after desired treatment conditions.
Nonapoptotic or nontreated cells should be included as negative 6.2.1 Inclusion dyes
controls. For combined intracellular staining, PBS should contain
0.1% azide to stop the cellular metabolism. For the dilution factors Inclusion dyes require intracellular enzymatic functionality in addition
of the staining buffers, distributor-recommended dilutions should be to the membrane integrity. Cytoplasmic esterases cleave the nonfluo-
applied and dilution optimization should be confirmed for each cell rescent molecules and yield fluorescent compounds. Examples of inclu-
type. sion dyes are fluorescein diacetate, carboxyfluorescein, and calcein
After washing step, 1,000,000–5,000,000 cells/condition or repli- (Johnson et al., 2013). Here, we describe an example protocol for cal-
cate are dissolved in 100 µl staining solution. For F2N12S staining, cein staining.
cells should be incubated for 5 min at room temperature. For annexin
V staining, cells should be incubated at 2–8◦ C for 30 min or 10–15 min Reagent preparation
at room temperature. During the incubation, cells should be protected Calcein powder is dissolved in cell culture grade DMSO to 5 mM. Small
from light. After incubation, cells are washed, and dissolved within aliquots of dissolved solution can be stored at 20◦ C. Like fluorometric
100–200 µl washing buffer or the buffer that is used to dilute the staining reagents, calcein is sensitive to buffers and solutions contain-
staining buffer. Emission signal is measured with the flow cytometer. ing esterases. It is not recommended to use intracellular fixation proto-
F2N12S signal can be obtained from the appropriate violet channels cols together with calcein dyes.
to collect the 585 and 530 nm emissions and for annexin V, appro-
priate channel is selected depending on the conjugated color (Dem- Protocol
chenko, 2013) (ThermoFisher Scientific). For gating strategies, see Cells are washed with PBS after desired treatment conditions. A total
Figure 6. of 1,000,000 cells are prepared for each replicate and condition. For
adherent cells, cells should be washed and stained prior to removing
Fixation the cells from the growth surface.
It is possible to fix annexin V-stained cells to do permeabilization for Stock calcein solution (5 mM) is diluted to 10 nM final concentra-
intracellular stainings or to perform further phenotypic stainings. The tion using culture medium. Cells are resuspended in the diluted cal-
fixation method should be alcohol free and aldehyde based, such as cein solution and incubated for 30 min at 37◦ C. Cells are washed twice
0.5–4% formaldehyde solution, diluted with PBS. For annexin V stain- after incubation and measured with a flow cytometer at excitation and
ing, it is very crucial that binding buffers or washing buffers do not emission wavelengths of 488 and 520 nm, respectively (Gillissen et al.,
contain Ca2+ chelators. For the fixation step, cells are washed after 2016). Percentage of the living cells can be calculated with one of the
annexin V staining to remove the unbound dyes. After washing, cells package software, such as FlowJo (FlowJo, LLC, USA), that is used to
are fixed with formaldehyde for 15–20 min at room temperature. After analyze the obtained Flow Cytometry Standard (FCS) files.
6.2.2 Exclusion dyes There are several commercially available assays targeting different
aspects of the mitochondria. JC-10 MMP kits are superior alternatives
Exclusion dyes are fluorescent compounds that are excluded from the of poor water-soluble JC-1 dye and can be found from Abcam, Merck,
living cells and accumulated in dying cells that have leaky membranes. and Sigma–Aldrich. In summary, JC-10 dye accumulates in mitochon-
Propidium iodide (PI) and 7-amino actinomycin D are well-known and dria matrix of healthy cells and gives a red emission signal at 570–
frequently used examples of exclusion dyes (Davey & Guyot, 2020; 590 nm. Once the cells are apoptotic or necrotic, JC-10 easily diffuses
Johnson et al., 2013). PI stains nucleic acids and live and dead cells can into the cytoplasm and becomes monomeric JC-10, which gives a green
be distinguished easily via very bright color of PI (Johnson et al., 2013). emission signal at 520–540 nm.
Here, we describe an example protocol for PI staining. MitoTracker dyes can be applied to measure total mitochondria
Reagent preparation mass or to study the changes in mitochondria mass following desired
PI powder is dissolved in PBS to 2 mg/ml. After dissolving, stock solu- treatments. MitoTracker Green FM from Cell Signaling and fromTher-
tion should be protected from light and can be stored at 4◦ C for short- moFisher Scientific are the examples of commercially available
term storage up to 1 month. MitoTracker assays. MitoTracker Green dye stains the mitochondria
of living cells but it is not dependent on the MMP. MitoTracker Green
Protocol
is not compatible with fixation and the signal can be obtained at
Cells suspensions are prepared after desired treatment conditions. A
excitation and emission wavelengths of 490 and 516 nm, respectively.
total of 1,000,000 cells/condition or 1,000,000 cells/replicate were
Another strategy of staining mitochondria is targeting mitochon-
resuspend in PBS containing 2 µg/ml PI final concentration (×1,000
drial permeability. This assay is provided by Abcam and ThermoFisher
dilution). Cells are incubated for 5–10 min, at dark and on ice. After
Scientific. The assays include a dye, calcein AM, and a quencher, CoCl2 ,
incubation, cells were measured with a flow cytometer without wash-
and both can diffuse into the cells easily. As described in the previous
ing. PI signal is obtained at 488 nm excitation/550 nm emission and
sections, calcein AM is cleaved by cytosolic esterases. After its cleav-
analyzed with a data analysis software (Johnson et al., 2013).
age, it is quenched by CoCl2 in cytoplasm, but retains the mitochon-
PI staining is not compatible with fixation.
dria of living healthy cells. Once the cells have mitochondrial damage
or apoptotic, mitochondria content is leaked and mitochondrial signal
6.2.3 Monomeric cyanine nucleic acid stains
is also lost.
Monomeric cyanine nucleic acid stains are highly sensitive nuclear

staining dyes targeting double-stranded nucleic acids. A group of 7 CONCLUSION
monomeric cyanine nucleic acid stains are provided by Invitrogen (part
of Life Technologies, USA). They are compatible with fixation steps and In this guideline, the most common cell viability assays applied in
are not excluded from the cellular membranes like other exclusion dyes. research labs, namely, dye exclusion, colorimetric, fluorometric, lumi-
Examples of monomeric cyanine nucleic acid stains are YO-PRO, TO- nometric, and flow cytometric assays, are presented. The mechanism
PRO, and JO-PRO (Invitrogen). Alternative dyes with a wide range of underlying each assay and the practice of viability assessment are dis-
emission/excision variation can be found. cussed in detail. Principles, advantages, and disadvantages of the cell
In addition, there are also commercially available fixable live/dead viability assays are summarized in Table 1. Ideally, a cell viability assay
staining kits that can be a good alternative for protocols including fixa- should be safe, rapid, reliable, efficient, and time- and cost-effective,
tion and intracellular staining. After fixation, the cells are dead so that and should not interfere with the test compound. Hence, while choos-
it is very important to start stainings first with live/dead dyes. Examples ing a cell viability assay, the mechanism of action of the test compound
can be listed as Zombie Cell Viability Dyes from Biolegend, LIVE/DEAD should be considered. Moreover, the type and origin of the cell line
Fixable Stains from ThermoFisher Scientific, Aqua Dead Cell Stain Kit also influence the performance of cell viability assays. Considering the
from Fisher Scientific, Live/Dead Fixable Staining Kit from Promocell, above, it can be concluded that the measurement of cell viability cannot
and so forth. be investigated satisfactorily by a single method. Therefore, it is recom-

mended that more than one assay should be applied for the evaluation
6.3 Mitochondria assays of the cell viability.
CONFLICT OF INTEREST
Mitochondria are the powerhouses of the cells. Membrane potential
The authors confirm that they have no conflict of interest to declare for
of mitochondria is very critical for energy synthesis and is well regu-
this publication. [Correction added on 14 June 2021, after first online
lated. Mitochondria population of a living cell is maintained by a pro-
publication: The ‘Conflict of interest’ section was added.]
cess called “biogenesis,” which is the balanced combination of fission
and fusion events and is a dynamic mechanism regulated according to
ORCID
the status of the cell (Sedlackova & Korolchuk, 2019). It is possible to
Senem Kamiloglu https://orcid.org/0000-0003-3902-4360
stain cells with dyes targeting mitochondria to understand the status
Gulce Sari https://orcid.org/0000-0002-8585-5889
of the cells such as apoptotic, high energy or mitochondria membrane
Esra Capanoglu https://orcid.org/0000-0003-0335-9433
potential (MMP) lost, and so forth.
346
TA B L E 1 Principles, advantages, and disadvantages of cell viability assays
Assay Principle Advantages Disadvantages References

Dye exclusion assays Determination of ∙ Simple ∙ Time-consuming and labor-intensive for large Kim et al., 2016; Krause,
(trypan blue, eosin, congo membrane integrity ∙ Rapid number of samples Carley, & Webb, 1984;
red, and erythrosine B) ∙ Inexpensive ∙ Only suitable for cell suspensions, that is, Ruben, 1988; Weisenthal,
∙ Versatile monolayers need trypsinization Dill, Kurnick, & Lippman,
∙ Require small number of cells ∙ Counting errors may occur due to poor cell 1983; Yip & Auersperg,
∙ Erythrosine B is nontoxic, does not bind dispersion, incorrect dilution, contamination of 1972; Aslantürk, 2018
to serum proteins, and does not require reusable counting chambers, air bubbles, or
incubation before counting inter-user variations
∙ Cannot differentiate between healthy cells and
viable cells that lost their function
∙ Trypan blue is toxic to mammalian cells
Colorimetric assays Determination of ∙ Simple ∙ MTT is not soluble in water, that is, additional Bopp & Lettieri, 2008; Cory
(MTT, MTS, XTT, WST-1, metabolic activity ∙ Inexpensive formazan dissolution step is required et al., 1991; Decker &
WST-8, LDH, SRB, and ∙ Safe ∙ MTT may interfere with cell culture medium Lohmann-Matthes, 1988;
NRU, CVS) ∙ Reproducible ∙ MTT is highly toxic to cells Fotakis & Timbrell, 2006;
∙ Precise ∙ Incubation time, cell type, and cell count may Skehan et al., 1990; Stone
∙ Accurate affect the measurements in MTS assay et al., 2009; Strober, 2015;
∙ Rapid ∙ Enzymatic regulation, pH, cellular ion Tominaga et al., 1999;
∙ Sensitive concentration, cell cycle variation, or other Präbst, Engelhardt,
∙ Reliable environmental factors may affect the Ringgeler, & Hübner, 2017;
∙ Applicable to both cell suspensions and measurements in XTT assay Aslantürk, 2018
adherent cells ∙ Changes in intracellular metabolic activity
∙ WST-1 and WST-8 are water soluble, having no direct effect on cell viability may affect
that is, no additional formazan the measurements in WST-8 assay
dissolution step is required ∙ Serum and some other compounds having
∙ WST-1 and WST-8 do not interfere with inherent LDH activity may affect the
phenol red or other culture mediums measurements in LDH assay
∙ WST-8 has low toxicity ∙ Cellular clumps or aggregates may affect the
measurements in SRB assay
∙ CVS assay is insensitive to changes in cell
metabolic activity
Fluorometric assays Nonspecific cleavage of a ∙ Simple ∙ Possible fluorescent interference caused by the Rotman & Papermaster,
(Resazurin [alamar blue] nonfluorescent ∙ Inexpensive applied test compounds 1966; Altman et al., 1993;
and 5-CFDA-AM) compound into a ∙ Safe ∙ Cannot be applied to newly thawed cells Larson et al., 1997;
fluorescent compound ∙ Rapid ∙ Light sensitive Schirmer et al., 1997;
by cellular esterases ∙ Applicable to a number of cell types Ganassin & Bols, 2000;
∙ Applicable to both cell suspensions and Schreer et al., 2005;
adherent cells Czekanska, 2011; Johnson
et al., 2013; Riss et al.,
2016
(Continues)
KAMILOGLU ET AL .
TA B L E 1 (Continued)
Assay Principle Advantages Disadvantages References

Luminometric assays Correlation between a ∙ Simple ∙ Assay is prone to results with technical errors McElroy, 1947; Bulich &
(ATP and real-time bioluminescent reaction ∙ Fast such as pipetting errors Isenberg, 1981; Ugarova
KAMILOGLU ET AL .
viability) and the effect of a tested ∙ Luminescent signal is quite stable ∙ The length of real-time viability assay et al., 1987; Voyevodina
compound ∙ Signal can be measured within a few measurement depends on the metabolic activity et al., 1990; Ugarova,
hours of the target cells; test compound can deplete 1993; Lundin, 2000;
∙ Very specific before the end point. To avoid it, maximum Lomakina et al., 2015; Riss
∙ Can be applied with very few cells (>50 incubation time is required to be optimized very et al., 2016; Aslantürk &
cells) carefully for each cell type Çelik, 2017
∙ Real-time viability assay is the only cell
viability method that allows to monitor
the cell viability in real time
Flow cytometric assays

Membrane asymmetry Detection of the ∙ Fixation is possible with Annexin V ∙ Annexin V staining is sensitive to buffers Demchenko, 2010, 2013
assays alterations in the staining containing Ca2+ chelators such as EDTA
(Annexin V and F2N12S composition of the outer ∙ F2N12S probe is a small molecule, not ∙ Light sensitive
probe) surface of cell membrane sensitive to proteases ∙ False positivity following trypsin treatment
∙ F2N12S staining is very fast (5 min)
∙ For both dyes, membrane
permeabilization is not required
∙ Both dyes can be combined with other
markers
Membrane permeability Determination of ∙ Exclusion dye propidium iodide has a very ∙ Apoptotic cells keep their membrane integrity Darzynkiewicz et al., 1992;
assays membrane integrity and bright color to distinguish live and dead and they are resistant to exclusion dyes Da Silveira et al., 2002;
(inclusion dyes, exclusion permeability cells ∙ Calcein (inclusion dyes) is sensitive to buffers Nagy et al., 2004; Johnson
dyes, and monomeric ∙ Monomeric cyanine nucleic acid stains containing esterases et al., 2013; Gillissen et al.,
cyanine nucleic acid are very specific double-stranded nucleic ∙ Calcein staining and propidium iodide staining 2016; Davey & Guyot,
stains) acid dyes cannot be combined with fixation protocols 2020
∙ Monomeric cyanine nucleic acid stains ∙ Light sensitive
are compatible with fixation protocols
Mitochondria assays Determination of ∙ Provide information about the energy ∙ MitoTracker Green is not compatible with Sedlackova & Korolchuk,
(JC-10, MitoTracker, and mitochondria membrane status of the cells in addition to viability fixation 2019;
mitochondria potential, mitochondria Commercial providers:
permeability assays) mass, or mitochondria Abcam, ThermoFisher
membrane permeability Scientific, Merch, and
Sigma–Aldrich
Abbreviations: CVS, crystal violet stain; F2N12S, 4′-N,N-diethylamino-6-(N-dodecyl-N-methyl-N-(3-sulfopropyl))ammoniomethyl-3-hydroxyflavone; LDH, lactate dehydrogenase; MTS, 3-(4,5-dimethylthiazol-
2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium; MTT, 3-[4,5-dimethylthiazol-2-yl]-2,5 diphenyl tetrazolium bromide; NRU, neutral red uptake; SRB, sulforhodamine B; WST-1, 2-(4-
iodophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfophenyl)-2H tetrazolium, monosodium salt; WST-8, 2-(2-methoxy-4-nitrophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfophenyl)-2H-tetrazolium, monosodium salt; XTT, 2,3-
bis-(2-methoxy-4-nitro-5-sulfophenyl)-2H-tetrazolium-5-carboxanilide; 5-CFDA-AM, 5-carboxyfluorescein diacetate acetoxymethyl ester.
347
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2020;1:332–349. https://doi.org/10.1002/fft2.44

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Received: 30 June 2020 Revised: 19 August 2020 Accepted: 27 August 2020

Guidelines for cell viability assays

Senem Kamiloglu1 Gulce Sari2 Tugba Ozdal3 Esra Capanoglu4

332 wileyonlinelibrary.com/journal/fft2 Food Frontiers. 2020;1:332–349.

There are several types of assays that can be used to deter-

3.1.1 Reagent preparation

FIGURE 2 Reduction of MTT to formazan crystals

Mean ODsample 3.2.2 Protocol

3.2.3 Calculation 3.4 WST-1 assay

aqueous solutions and directly quantified using a scanning multiplate

is quantified spectrophotometrically utilizing an ELISA plate test. An

The percentage of cell viability is calculated using the following equa-

3.3.3 Calculation cells produces an orange-colored formazan product, which is directly

tion: is found to be more sensitive for cell viability measurements than

3.6.1 Reagent preparation

3.7 SRB assay 3.8 NRU assay

3.9 CVS assay 4 FLUOROMETRIC ASSAYS

4.1 Resazurin (alamar blue) assay

Monomeric cyanine nucleic acid stains are highly sensitive nuclear

and so forth. be investigated satisfactorily by a single method. Therefore, it is recom-

TA B L E 1 Principles, advantages, and disadvantages of cell viability assays

Assay Principle Advantages Disadvantages References

Assay Principle Advantages Disadvantages References

Flow cytometric assays

REFERENCES De Jong, D. W., & Woodlief, W. G. (1977). Fluorimetric assay of tobacco

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