In vitro assessment of cellular viability has become a generic approach in addressing a vast range of biological questions in many areas of biomedical research. Cell viability assays are often used to screen collections of compounds to determine if the test molecules have effects on cell proliferation or show direct cytotoxic effects that eventually lead to cell death. According to the number and type of cells used, choosing a proper assay method to decided whether the expected outcome is valid or not.

1. Comparison of Different
Methods to Measure Cell
Viability
In vitro assessment of cellular viability has become a generic approach in addressing a
vast range of biological questions in many areas of biomedical research. Cell viability
assays are often used to screen collections of compounds to determine if the test
molecules have effects on cell proliferation or show direct cytotoxic effects that eventually
lead to cell death. According to the number and type of cells used, choosing a proper
assay method to decide whether the expected outcome is valid or not.
Viable cells can convert the substrate into product resulting in a colored or fluorescent
product that can be detected with a plate reader, and the signal is directly proportional to
the number of viable cells present. But the dying cells lose the conversion ability, that
difference provides the basis for many of the commonly used cell viability assays. The
tetrazolium reduction, resazurin reduction, and protease activity assays all take
advantage of this theory. The ATP assay is somewhat different in that the addition of
assay reagent immediately ruptures the cells, thus there is no incubation period of the
reagent with a viable cell population.
MTT Tetrazolium Assay
Principle
The MTT tetrazolium reduction assay was the first homogeneous cell viability assay
developed for a 96-well format that was suitable for high throughput screening (HTS).
NAD(P)H-dependent cellular oxidoreductase enzymes in viable cells can reduce the
tetrazolium dye MTT 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide to its
insoluble formazan, which has a purple color. The quantity of formazan is measured by
recording changes in absorbance at 570nm using a spectrophotometer. Under defined
conditions, it can reflect the number of viable cells present. MTT assays can be used to
measure cytotoxicity (loss of viable cells) or cytostatic activity (shift from proliferation to
quiescence) of potential medicinal agents and toxic materials.

2. Figure 1. The process of the MTT
colorimetric assay.
Figure 2. A: Structures of MTT and colored formazan product. B:
Direct correlation of formazan absorbance with B9 hybridoma cell number and
time-dependent increase in absorbance (Riss, T. L. 2004).
Advantages
• Fewer steps, uses fewer materials, and does not carry the added burden of radioactive
waste disposal.
Disadvantages
• Not suitable for suspending cells.
• Must be optimized for seeding amount and assay duration to obtain satisfactory results.
• Precipitated protein and cellular debris present in the culture plate wells are a potential
source of experimental error because they interfere with the optical readings.
MTS Tetrazolium Assay
Principle
MTS [3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-
tetrazolium] is a new kind of tetrazolium salts, it can be reduced by viable cells
to generate formazan products that are directly soluble in cell culture medium. For

3. these improved tetrazolium reagents, the usual protocol is simply to add them to the
culture medium and read the absorption of the reduced product at an appropriate time.
This approach eliminates a liquid handling step during the assay procedure; thus, it
obviously saves time and eliminates potential errors such as cell loss that can occur
when removing culture medium and subsequently solubilizing cells.
Figure 3. a: Intermediate electron acceptor pheazine ethyl sylfate (PES) transfers
electron from NADH in the cytoplasm to reduce MTS in the culture medium into an
aqueous soluble formazan. b: MTS assay for cell viability was performed with increasing
concentration of different peptides at different time courses. (Alhoot, 2013)
Advantages
• Simple operation. MTS is more efficient than MTT and produces water-soluble
formazan the does not require DMSO dissolution.
• Due to the MTS produce darker formazan product, the absorbance value range is more
sensitive and accurate, as well as it's easier to judge positive results.
• Fast. 2-3 hours are enough for reaction while the MTT need 4 hours.
• Good repeatability.
Disadvantages
• The MTS regents is more expensive than MTT.
CCK-8 (WST-8) Assay
Principle
As a second-generation water-soluble tetrazolium salt, WST-8 [2-(2-methoxy-4-
nitrophenyl)-3-(4-nitrophenyl)- 5-(2,4-disulfophenyl)-2H-tetrazolium, monosodium salt]
has better soluble than MTS. WST-8 is reduced by dehydrogenases in cells to give an
orange colored product (formazan), which is soluble in the tissue culture medium. And
the amount of the formazan dye generated by dehydrogenases in cells is directly
proportional to the number of living cells.

4. Figure 4. A: Structures of WST-8 and WST-8 formazan. B:
Principle of the cell viability detection with Cell Counting Kit-8.
Figure 5. The procedure of CCK-8 assay.
Advantages
• CCK-8 is a one-bottle solution; no premixing of components is required.
• CCK-8 is simple, rapid, low consumed and sensitive with repeatability.
• CCK-8, being nonradioactive, allows sensitive colorimetric assays for the determination
of the number of viable cells in cell proliferation and cytotoxicity assays.
Disadvantages
• More expensive than MTT regents
• the color of CCK-8 is closed to the medium with phenol red which may miss adding
regents if not careful. The operation mistake will disturb the data result.
Sulforhodamine B colorimetric assay
Principle

5. The sulforhodamine B (SRB) assay was developed in 1990, one of the most widely used
methods for in vitro cytotoxicity screening. The assay relies on the ability of SRB to bind
to protein components of cells: SRB is a bright-pink aminoxanthene dye with two sulfonic
groups that bind to basic amino-acid residues components of cells under mildly acidic
conditions, and dissociate under basic conditions. As the binding of SRB is
stoichiometric, the amount of dye extracted from stained cells can be used as a proxy for
cell mass directly, and the amount of bound dye is proportional to the cell mass.
Figure 6. Sulforhodamine B colorimetric assay in cell culture to analyze cell
proliferation. A. Cell number titration (H441) using the SRB assay 384-well
format, n = 6, error bars = SD. B. miR-34a dose response analysis in lung cancer
cell lines H441 and H358 using SRB 96-well format, n = 3. (Vichai, V, 2006)
Advantages
• Because the cell was fixed, SRB staining rarely affected by other compounds interfere
which can remain for a long time.
• SRB soluble with Tris-based solution is also stable for a long time. Therefore, the 96-
hole cell culture plate at different time points can be determined at the same time.
Disadvantages
• The application of the SRB assay is limited to manual or semiautomatic screening due
to the multiple washing and drying steps, which are not amenable to automation.
• The operation procedure is complicated.
Conclusion
MTT MTS CCK-8 SRB

6. Solubility Indissolva
ble
Water solu
ble
Water solu
ble
Indissolva
ble
Detection Wavele
ngth
490nm 450nm 450nm 510nm
Character Solid Liquid Liquid Liquid
Usage Prepare
the
solutions
Use it right
after it was
ready
No need to
prepare
Prepared
beforehan
d
Need to redissolve
or not
Yes, by
DMSO
No No Yes, by
Tris-base
solution
Convenience ++ +++ +++ +
Detection speed + ++ +++ +
Repeatability + ++ +++ ++
Stability + + ++ +++
Related Products & Services
Cell Viability Assays
Cell Proliferation Kit (MTT)
MTS Cell Proliferation Colorimetric Assay Kit
BrdU Cell Proliferation Assay Kit
BrdU Staining Kit for Flow Cytometry FITC
References:
1. Riss, T. L.; et al. Assay guidance manual. Sittampalam, GS, et al (2004).
2. Alhoot, M. A.; et al. Inhibition of dengue virus entry into target cells using synthetic
antiviral peptides. International journal of medical sciences. 2013, 10(6), 719.
3. Vichai, V.; et al. (2006). Sulforhodamine B colorimetric assay for cytotoxicity
screening. Nat Protoc. 2006, 1(3): 1112-1116.