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Protocol

Analysis of Cell Viability by the MTT Assay

Priti Kumar, Arvindhan Nagarajan, and Pradeep D. Uchil

Among viability assays that depend on the conversion of substrate to chromogenic product by live cells,
the MTT assay is still among one of the most versatile and popular assays. The MTT assay involves the
conversion of the water-soluble yellow dye MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazo-
lium bromide] to an insoluble purple formazan by the action of mitochondrial reductase. Formazan is
then solubilized and the concentration determined by optical density at 570 nm. The result is a
sensitive assay with excellent linearity up to
106 cells per well. As with the alamarBlue assay, small
changes in metabolic activity can generate large changes in MTT, allowing one to detect cell stress upon
exposure to a toxic agent in the absence of direct cell death. The assay has been standardized for
adherent or nonadherent cells grown in multiple wells. The protocol uses a standard 96-well plate. This
can be scaled up, however, to suit a different plate format. Plate 500–10,000 cells per well in a 96-well
plate. The assay has good linearity up to 106 cells.

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 material 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
Cultured mammalian cells that have been transfected
Most mammalian cell types reduce the tetrazolium salt sufficiently to perform MTT assays accurately at low cell
numbers. However, it may be necessary to increase the cell number to 1 × 105 to 5 × 105/mL when using blood
cells to obtain a significant 570-nm absorbance reading (Chen et al. 1990). The optimum number of cells for the
assay may also be determined empirically by performing the assay with varying numbers of cells in triplicate wells.

DMSO
MTT stock solution (12 mM) <R>
SDS–HCl solution (10%) <R>
This solution is needed for dissolving the insoluble formazan product formed at the end of the reaction. Alterna-
tively, the formazan product can be dissolved in dimethyl sulfoxide (DMSO).

Tissue culture medium

Equipment
Plates (96 well), standard flat-bottom wells
Plates (96 well), either V or round bottom
Spectrophotometer with multiwell tissue culture plate reading ability

From the Molecular Cloning collection, edited by Michael R. Green and Joseph Sambrook.
© 2018 Cold Spring Harbor Laboratory Press
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469
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P. Kumar et al.

Reduced form N N
N Electron mediator H
N
+ N N
N N S S
CH3 CH3
Br–
N N
CH3 CH3
MTT MTT formazan

FIGURE 1. Reduction of MTT to MTT formazan.

METHOD

The MTT assay was developed by Mossman (1983), and the reaction is illustrated in Figure 1.

Labeling Cells
1. For adherent cells, remove the medium, and replace it with 100 µL of fresh culture medium. For
nonadherent cells, centrifuge the microplate, pellet the cells, carefully remove as much medium as
possible, and replace it with 100 µL of fresh medium.
Phenol Red indicator in cell culture medium can interfere with the MTT reaction. It may be necessary to
grow the cells in an indicator-free medium or replace it with an indicator-free medium just before the
addition of MTT reagent.
2. Add 10 µL of the 12 mM MTT stock solution to each well. Include a negative control of 10 µL of
the MTT stock solution added to 100 µL of medium alone.
3. Incubate the plate for 4 h at 37˚C. At high cell densities (>100,000 cells per well), the incubation
time can be shortened to 2 h. Solubilize the formazan produced using either SDS–HCl or DMSO.
Several companies market the MTT assay kit in its original format. Some kits (e.g., Promega’s CellTiter 96
Aqueous) use MTS instead of MTT as a substrate. The MTS tetrazolium is similar to the MTT tetrazolium,
with the advantage that the formazan product of MTS reduction is soluble in cell culture medium and does
not require a solubilization step in the assay protocol.

Solubilizing Formazan with SDS-HCl

4. Add 100 µL of SDS–HCl solution to each well and mix thoroughly using the pipette. This
dissolves the formazan produced.
5. Incubate the microplate for 4–18 h at 37˚C in a humidified chamber depending on the amount of
formazan product formed. Longer incubations will decrease the sensitivity of the assay.
6. Mix each sample again using a pipette, and read the absorbance at 570 nm.

Solubilizing Formazan with DMSO

A quicker and more popular solubilization reagent than SDS–HCl is DMSO.

7. Remove all but 25 µL of medium from the wells.

For nonadherent cells, it may be necessary to first centrifuge the plates to sediment the cells.

8. Add 50 µL of DMSO to each well and mix thoroughly with a pipette.

9. Incubate the plates for 10 min at 37˚C.
10. Mix each sample again, and read the absorbance at 540 nm.

DISCUSSION

To calculate the percentage of dead cells, use a positive control of 100% lysed cells. Cells are lysed by
freeze–thaw and pipetting before addition of the MTT. The average of the absorbance values for the

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The MTT Assay

0.60

0.50

Corrected absorbance, 570 nm

0.40

0.30
IC50 = 0.016 ng/mL
0.20

0.10

0.00
0 0.003 0.012 0.048 0.195 0.78
Toxic agent (ng/mL)

FIGURE 2. A sample plot for determining the IC50 of a toxic agent. (Based on an assay for TNF-α effects on L929 cells,
modified with permission from Promega Corp.)

positive control (100% lysed cells) is used as a blank value and subtracted from all other absorbance
values to yield the corrected absorbance values. Plot the corrected absorbance values 570 nm (on the
ordinate) versus concentration of cytotoxic agent (abscissa, log scale) as shown in Figure 2, and
determine the IC50 value by locating the abscissa value corresponding to one-half the maximum
absorbance value (IC50 is the inhibitory concentration of cytotoxic agent necessary to kill one-half of
the cell population).

RECIPES

MTT Stock Solution (12 mM)

MTT 5 mg
PBS 1 mL
Prepare a 12 mM stock solution by dissolving 5 mg of MTT [3-(4,5-dimethylthiazol-2-
yl)-2,5-diphenyltetrazolium bromide] in 1 mL of PBS. This stock solution will be
enough for 100 reactions (10 µL per reaction). Once prepared, the MTT solution can
be stored for 4 wk at 4˚C protected from light.

SDS–HCl Solution (10%)

SDS 1g
HCl (0.01 N) 10 mL
Prepare by dissolving 1 g of SDS in 10 mL of 0.01 N HCl. Mix the solution by vortexing
until the SDS completely dissolves. One milliliter is sufficient for 100 reactions (10 µL
per reaction).

REFERENCES

Chen C-H, Campbell PA, Newman LS. 1990. MTT colorimetric assay Mossman T. 1983. Rapid colorimetric assay for cellular growth and surviv-
detects mitogen responses of spleen but not blood lymphocytes. Int als: Application to proliferation and cytotoxicity assays. J Immunol
Arch Allergy Appl Immunol 93: 249–255. Methods 65: 55–63.

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Analysis of Cell Viability by the MTT Assay

Priti Kumar, Arvindhan Nagarajan and Pradeep D. Uchil

Cold Spring Harb Protoc; doi: 10.1101/pdb.prot095505

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