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Protocol

Crystal Violet Assay for Determining Viability of Cultured Cells

Maria Feoktistova,1 Peter Geserick,1 and Martin Leverkus1,2,3
1
Section of Molecular Dermatology, Department of Dermatology, Venereology and Allergology, Medical Faculty
Mannheim, University of Heidelberg, Heidelberg 68167, Germany; 2Department of Dermatology & Allergology,
University Hospital of RWTH Aachen University, 52074 Aachen, Germany

Adherent cells detach from cell culture plates during cell death. This characteristic can be used for the
indirect quantification of cell death and to determine differences in proliferation upon stimulation
with death-inducing agents. One simple method to detect maintained adherence of cells is the staining
of attached cells with crystal violet dye, which binds to proteins and DNA. Cells that undergo cell death
lose their adherence and are subsequently lost from the population of cells, reducing the amount of
crystal violet staining in a culture. This protocol describes a quick and reliable screening method that is
suitable for the examination of the impact of chemotherapeutics or other compounds on cell survival
and growth inhibition. However, characterization of the cause of reduced crystal violet staining
requires additional methods detailed elsewhere.

MATERIALS

It is essential that you consult the appropriate Material Safety Data Sheets and your institution's Environmental
Health and Safety Office for proper handling of equipment and hazardous materials used in this protocol.
RECIPES: Please see the end of this protocol for recipes indicated by <R>. Additional recipes can be found online at
http://cshprotocols.cshlp.org/site/recipes.

Reagents
Adherent cell line of interest
Cell culture medium
Use the appropriate cell culture medium according to the cell type/line being cultured.

Crystal violet staining solution (0.5%) <R>

Drug(s) appropriate for experimental goals (e.g., CD95L, poly[I:C])
Methanol
Equipment
96-well plate reader that is able to read absorbance at 570 nm (e.g., VICTOR3 Multilabel Plate Counter
from PerkinElmer)
96-well tissue culture plates
Bench rocker (2D or 3D)
Filter paper
Incubator (37˚C, 5% CO2, humidified)
Vacuum aspirator

3
Correspondence: mleverkus@ukaachen.de
© 2016 Cold Spring Harbor Laboratory Press
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M. Feoktistova et al.

METHOD

See Figure 1 for an overview of the crystal violet assay. Here, we describe the method using a 96-well format. However,
the same method can be used with other plate sizes by simply scaling cell numbers and volumes accordingly. Perform
Steps 1–3 under a laminar flow hood.
1. Seed cells in a 96-well plate. To three wells, add medium without cells. Ensure that the volume of
the culture medium is ≥100 µL/well to avoid evaporation effects. Incubate the cells for 18–24 h at
37˚C to enable adhesion of cells to wells.
Usually 1–2 × 10 4 cells/well should be seeded to achieve confluence of 40%–50%. Initial cell confluence
depends on the proliferation and cell size of the cell line used; therefore adjust accordingly. The wells
without cells will serve as controls for nonspecific binding of the crystal violet dye.

2. Aspirate the medium from the wells, and add ≥100 µL/well of fresh medium supplemented with
drugs appropriate for the experimental goals (e.g., 2–20 µg/mL of poly[I:C] for HaCaT cells). Do
not let the cells dry out when changing medium; cells that have dried out will not undergo cell
death induction. Treat cells in triplicate wells for each condition. Do not treat cells in three of
the wells (these will serve as controls). Incubate cells for the desired time in the desired conditions
(e.g., in case of HaCaT and poly(I:C) treatment it is for 18–24 h at 37˚C).
Different concentrations of other stimulants (e.g., death ligands such as TRAIL [1–1000 ng/mL] or TNF
[1–1000 ng/mL]) should be analyzed in preliminary experiments to determine dose-dependent cell death
induction before co- and prestimulation with respective inhibitors (e.g., caspase inhibitor: zVAD-fmk
[10 µM]; IAP antagonist: GT12911e [100 nM]).

3. Aspirate the medium, and wash the cells twice in a gentle stream of tap water. Tilt the plate to
prevent the stream of water from hitting the cell monolayer directly. After the wells have filled
with water, immediately aspirate the water from the wells. After washing, invert the plate on filter
paper and tap the plate gently to remove any remaining liquid.

Control Stimulus

Seed cells

Stimulate cells

Add crystal violet dye

Wash four times with water

Add methanol

Measure OD at 570 nm
% Surviving attached cells
(% crystal violet OD)

Perform statistical analysis

Control Stimulus
FIGURE 1. Schematic workflow for crystal violet assay.

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Crystal Violet Assay

Some cells (e.g., keratinocytes) adhere strongly to the surface of cell culture plates (Boukamp et al. 1988),
whereas others (e.g., HeLa or 293T cells) adhere weakly and are, therefore, easily flushed off the plate
surface during washing. Although the washing step increases the sensitivity of the assay, it can be skipped for
cells that do not adhere well to the plate.

4. Add 50 µL of 0.5% crystal violet staining solution to each well, and incubate for 20 min at room
temperature on a bench rocker with a frequency of 20 oscillations per minute.
5. Wash the plate four times in a stream of tap water as described in Step 3. After washing, invert the
plate on filter paper and tap the plate gently to remove any remaining liquid. Air-dry the plate
without its lid for at least 2 h at room temperature.
It is recommended to dry the plate at room temperature for 16–24 h.

6. Add 200 µL of methanol to each well, and incubate the plate with its lid on for 20 min at room
temperature on a bench rocker with a frequency of 20 oscillations per minute.
7. Measure the optical density of each well at 570 nm (OD570) with a plate reader.
8. Subtract the average OD570 of the wells without cells from the OD570 of each well on the plate.
The OD570 of the wells without cells, but processed identically to wells with cells, defines the background
of the staining method.

9. Set the average OD570 of nonstimulated cells to 100%. Then determine the percentage of
stimulated cells that are viable (attached) by comparing the average OD570 values of stimulated
cells with the OD570 values of the nonstimulated cells.
See Troubleshooting.

10. Calculate the mean and the standard error of the mean for at least three independent experiments.

TROUBLESHOOTING

Problem (Step 9): Wells treated with the same conditions stain unequally with crystal violet.
Solution: Before plating adherent cells, make sure that the cells are in a homogenous, single-cell
suspension. If they are not, clumps of cells can be seeded in wells, resulting in unequal cell
densities. After crystal violet staining, this will give increase to OD570 measurements with large
standard errors that inaccurately reflect loss of viability.

DISCUSSION

Crystal violet staining is a quick and versatile assay for screening cell viability under diverse stimu-
lation conditions (Geserick et al. 2009). However, it is potentially compromised by proliferative
responses that occur at the same time as cell death responses. Therefore, chemical inhibitors of
caspases and/or of necroptosis may be incorporated into the assay (Degterev et al. 2008; Sun et al.
2012). Alternatively, molecular studies (e.g., overexpression or knockdown) can be performed to
more specifically address the nature of cell death (Feoktistova et al. 2011).

RECIPE

Crystal Violet Staining Solution (0.5%)

0.5 g crystal violet powder (Sigma-Aldrich)

80 mL distilled H2O
20 mL methanol
Dissolve crystal violet powder in H2O and then add methanol. No sterilization procedures are
required. Store solution in the dark at room temperature. Use within 2 mo.

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M. Feoktistova et al.

ACKNOWLEDGMENTS

We thank all current and previous members of the Section of Molecular Dermatology, Medical
Faculty of Mannheim, University of Heidelberg for their discussions and suggestions. The work in
the laboratory of M.L. was supported by grants from the Deutsche Forschungsgemeinshaft (Le953/
5-1, 6-1, 8-1), the Wilhelm-Sander-Stiftung (2008.072.1), and the Mildred-Scheel-Stiftung (Projekt
109891).

REFERENCES

Boukamp P, Petrussevska RT, Breitkreutz D, Hornung J, Markham A,
Fusenig NE. 1988. Normal keratinization in a spontaneously immor-
talized aneuploid human keratinocyte cell line. J Cell Biol 106: 761–771.
Degterev A, Hitomi J, Germscheid M, Ch’en IL, Korkina O, Teng X, Abbott D,
Cuny GD, Yuan C, Wagner G, et al. 2008. Identification of RIP1 kinase as
a specific cellular target of necrostatins. Nat Chem Biol 4: 313–321.
Feoktistova M, Geserick P, Kellert B, Dimitrova DP, Langlais C, Hupe M,
Cain K, MacFarlane M, Hacker G, Leverkus M. 2011. cIAPs block
Ripoptosome formation, a RIP1/caspase-8 containing intracellular
cell death complex differentially regulated by cFLIP isoforms. Mol
Cell 43: 449–463.
Geserick P, Hupe M, Moulin M, Wong WW, Feoktistova M, Kellert B,
Gollnick H, Silke J, Leverkus M. 2009. Cellular IAPs inhibit a cryptic
CD95-induced cell death by limiting RIP1 kinase recruitment. J Cell
Biol 187: 1037–1054.
Sun L, Wang H, Wang Z, He S, Chen S, Liao D, Wang L, Yan J, Liu W, Lei X,
et al. 2012. Mixed lineage kinase domain-like protein mediates necrosis
signaling downstream of RIP3 kinase. Cell 148: 213–227.

346 Cite this protocol as Cold Spring Harb Protoc; doi:10.1101/pdb.prot087379

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Cold Spring Harbor Laboratory Press

Crystal Violet Assay for Determining Viability of Cultured Cells

Maria Feoktistova, Peter Geserick and Martin Leverkus

Cold Spring Harb Protoc; doi: 10.1101/pdb.prot087379

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