Cell Viability and Proliferation Assays

By: Article Based on, BioFiles v6 n5, 17–21


Assays to measure cellular proliferation, cell viability, and cytotoxicity are commonly used to monitor the response and health of cells in culture after treatment with various stimuli. The proper choice of an assay method depends on the number and type of cells used as well as the expected outcome. Assays for cell proliferation may monitor the number of cells over time, the number of cellular divisions, metabolic activity, or DNA synthesis. Cell counting using viability dyes such as trypan blue or Calcein-AM can provide both the rate of proliferation as well as the percentage of viable cells.

Overview of Cell Viability and Proliferation Assays

Name Overview Detection Method Advantage Disadvantage
BrdU Assay BrdU incorporates
into newly synthesized
DNA and detected
using anti-Brdu
ICC, IHC, FACS, ELISA Cell Cycle Kinetics
Single Cell Resolution
Lengthy Protocol
Potential DNA Damage
EdU Assay Similar to BrdU
technique but uses
Click-Chemistry detection
without antibodies
ICC, IHC, FACS, ELISA Less Toxic than BrdU
Faster Protocol No DNA Denaturation
Expensive Reagents
MTT Assay MTT, a yellow tetrazole,
is reduced to purple formazan in living
Spectrophotometer Fast Protocol
High Throughput
Endpoint Assay
Overestimation of Viability
Final Solubilization Step
XTT Assay Actively respiring
cells convert the
XTT to a water-
soluble, orange
colored formazan
Spectrophotometer High Sensitivity
Large Dynamic Range
Water Soluble
Endpoint Assay
Overestimation of Viability
WST-1 Assay WST-1 is cleaved to
a soluble formazan
by a complex cellular
mechanism that
occurs primarily at
the cell surface.
Spectrophotometer Highest Sensitivity
Faster Protocol
Endpoint Assay
Overestimation of Viability
Ki67 Antibodies to Ki-67
nuclear protein can
be used to measure
cellular proliferation.
ICC, IHC, WB In Vivo Applications Difficult to Quantify
Requires Fixation
non-fluorescent cell
permeable dye, is
cleaved by
intracellular esterases
which results in the
green fluorescence.
ICC, FACS Live Cell Analysis
Long Experiments
Compatible with Lymphocytes
Green Channel Emission
Live/Dead Assays Simultaneous
fluorescence staining
of viable and dead
cells using calcein-AM
and propidium
iodide (PI), which
stain viable and
dead cells,
ICC, FACS Live Cell Analysis
Rapid Protocol
Single Cell Resolution
Background Autofluorecnce
Trypan Blue Dye exclusion test is
based upon the
concept that viable
cells do not take up
impermeable dyes
but dead cells are
permeable and take
up the dye.
Microscopy Low Cost
Rapid Protocol
β-gal Beta-galactosidase
(β-Gal) enzyme
activity is detectable
in senescent cells
that do not
IHC Low Cost
Rapid Protocol
Difficult to Quantify
Endpoint Assay

DNA Synthesis Proliferation Assays

BrdU Cell Proliferation Assays

Cell proliferation may be studied by monitoring the incorporation of a radioisotope, [3H]-thymidine, into cellular DNA, followed by autoradiography. Alternatively, 5-bromo-2′-deoxy-uridine (BrdU assays) may be used instead of thymidine. Cells that have incorporated BrdU into DNA are easily detected using a monoclonal antibody against BrdU and an enzyme- or fluorochrome-conjugated second antibody.

BrdU Cell Proliferation Assays

Figure 1. A. Proliferating cells in the eye of E4 chick embryo using BrdU staining B. Anti-BrdU antibody validation using Camptothecin. By treating Jurkat cells with cell cycle arrest agent Camptothecin, circulating cells are trapped at the G1/S phase transition.


EdU Proliferation Assays

Baseclick EdU proliferation assays provide an efficient method for fluorescence detection of replicating DNA. The modified nucleoside EdU is added to live cells and is incorporated into replicating DNA. Cu-induced click chemistry allows rapid attachment of fluorescent probes to the EdU. This provides for a quantitative way to monitor cells that are proliferating. The assays are available in various formats for microscopy imaging, flow cytometry, high throughput screening, and for in vivo experiments. Four different fluorescent probes with excitation near 488, 555, 594, and 647 are available so that the readout can be multiplexed with other fluorescent probes.

Edu-Click cell proliferation kits

Figure 2. Edu-Click cell proliferation kits incorporates EdU (5-ethynyl-2’-deoxyuridine) into DNA during active DNA synthesis and is measured using a click-chemistry fluorescent detection method.


Metabolic Proliferation Assays

Assays that measure metabolic activity are suitable for analyzing proliferation, viability, and cytotoxicity. The reduction of tetrazolium salts such as MTT, XTT, WST-1 to colored formazan compounds or the bioreduction of resazurin only occurs in metabolically active cells. Actively proliferating cells increase their metabolic activity while cells exposed to toxins will have decreased activity.

MTT Cell Proliferation Assays

MTT (3-[4,5-dimethylthiazol-2-yl]-2,5- diphenyltetrazolium bromide; thiazolyl blue) is a water soluble tetrazolium salt yielding a yellowish solution when prepared in media or salt solutions lacking phenol red. Dissolved MTT is converted to an insoluble purple formazan by cleavage of the tetrazolium ring by dehydrogenase enzymes. This water insoluble formazan can be solubilized using isopropanol or other solvents and the dissolved material is measured spectrophotometrically yielding absorbance as a function of concentration of converted dye.

XTT Cell Proliferation Assays

In contrast to MTT, the cleavage product of XTT is soluble in water; therefore, a solubilization step is not required. The tetrazolium salt XTT is cleaved to formazan by a complex cellular mechanism. This bioreduction occurs in viable cells only, and is related to NAD(P)H production through glycolysis. Therefore, the amount of formazan dye formed directly correlates to the number of metabolically active cells in the culture.

WST-1 Cell Proliferation Assays

The stable tetrazolium salt WST-1 is cleaved to a soluble formazan by a complex cellular mechanism that occurs primarily at the cell surface. This bioreduction is largely dependent on the glycolytic production of NAD(P)H in viable cells. Therefore, the amount of formazan dye formed directly correlates to the number of metabolically active cells in the culture.

The MTT assay is a colorimetric assay for assessing cell proliferation based on metabolic activity

                                                          Yellow                                                   Purple


Figure 3. The MTT assay is a colorimetric assay for assessing cell proliferation based on metabolic activity. NAD(P)H-dependent cellular oxidoreductase enzymes reflect the number of viable cells present. These enzymes are capable of reducing the yellow tetrazolium dye MTT 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide to its insoluble formazan, which has a purple color.


Fluorescent Dye Proliferation Assays

CFSE Labeling

5(6)-Carboxyfluorescein diacetate N-succinimidyl ester (CFSE) is a popular choice for measuring the number of cellular divisions a population has undergone. Upon entering the cell, CFSE is cleaved by intracellular esterases to form the fluorescent compound and the succinimidyl ester group covalently reacts with primary amines on intracellular proteins. Upon division, the fluorescence intensity of each daughter cell is halved which allows for the simple detection of the number of cell divisions by flow cytometry. CFSE has been widely used to measure the proliferation of lymphocytes and t-cells.

Live/Dead Cell Double Staining

Live/Dead Cell Double Staining can be utilized for simultaneous fluorescence staining of viable and dead cells. Calcein-AM is a highly lipophilic and cell membrane permeable dye. Though Calcein-AM itself is not a fluorescent molecule, the calcein generated from Calcein-AM by esterase in a viable cell emits a strong green fluorescence (λex 490 nm, λem 515 nm). Therefore, calcein-AM only stains viable cells. Alternatively, the nuclei staining dye Propidium Iodine cannot pass through a viable cell membrane. It reaches the nucleus by passing through disordered areas of dead cell membrane, and intercalates with the DNA double helix of the cell to emit red fluorescence (λex 535 nm, λem 617 nm). Since both Calcein and PI-DNA can be excited with 490 nm light, simultaneous monitoring of viable and dead cells is possible with a fluorescence microscope.

Trypan Blue Viability Assays

Trypan Blue Exclusion

Trypan Blue is one of several stains recommended for use in dye exclusion procedures for viable cell counting. This method is based on the principle that live (viable) cells do not take up certain dyes, whereas dead (non-viable) cells do. Staining facilitates the visualization of cell morphology.

NOTE: Trypan Blue has a greater affinity for serum proteins than for cellular protein. If the background is too dark, cells should be pelleted and resuspended in protein-free medium or salt solution prior to counting.

Trypan Blue Protocol

  1. Prepare a cell suspension in a balanced salt solution (e.g., Hanks' Balanced Salts [HBSS], Cat. No. H9269).
  2. Transfer 0.5 ml of 0.4% Trypan Blue solution (w/v) to a test tube. Add 0.3 ml of HBSS and 0.2 ml of the cell suspension (dilution factor = 5) and mix thoroughly. Allow to stand for 5 to 15 minutes.
    Note: If cells are exposed to Trypan Blue for extended periods of time, viable cells, as well as non-viable cells, may begin to take up dye.
  3. With the cover-slip in place, use a Pasteur pipette or other suitable device to transfer a small amount of Trypan Blue-cell suspension mixture to both chambers of the hemacytometer. Carefully touch the edge of the cover-slip with the pipette tip and allow each chamber to fill by capillary action. Do not overfill or underfill the chambers.
  4. Starting with chamber 1 of the hemacytometer, count all the cells in the 1 mm center square and four 1 mm corner squares. Non-viable cells will stain blue. Keep a separate count of viable and non-viable cells.
    Note: Count cells on top and left touching middle line of the perimeter of each square. Do not count cells touching the middle line at bottom and right sides.
  5. Repeat this procedure for chamber 2.
    Note: If greater than 10% of the cells appear clustered, repeat entire procedure making sure the cells are dispersed by vigorous pipetting in the original cell suspension as well as the Trypan Blue-cell suspension mixture. If less than 200 or greater than 500 cells (i.e., 20–50 cells/square) are observed in the 10 squares, repeat the procedure adjusting to an appropriate dilution factor.
  6. Withdraw a second sample and repeat count procedure to ensure accuracy.


Cell Counts – Each square of the hemacytometer, with cover-slip in place, represents a total volume of 0.1 mm3 or 10-4 cm3. Since 1 cm3 is equivalent to approximately 1 ml, the subsequent cell concentration per ml (and the total number of cells) will be determined using the following calculations:

Cells Per mL = the average count per square × dilution factor × 10(count 10 squares)

Example: If the average count per square is 45 cells × 5 × 104 = 2.25 × 106 cells/ml.

Total Cells = cells per ml × the original volume of fluid from which cell sample was removed.

Example: 2.25 × 106 (cells/ml) × 10 ml (original volume) = 2.25 × 107 total cells.

Cell Viability (%) = total viable cells (unstained) ÷ total cells (stained and unstained) × 100.

Example: If the average count per square of unstained (viable) cells is 37.5, the total viable cells = [37.5 × 5 × 104] viable cells/ml × 10 ml (original volume) = 1.875 × 107 viable cells. Cell viability (%) = 1.875 × 107 (viable cells) ÷ 2.25 × 107 (total cells) × 100 = 83% viability.

Counting Cells Using Hemocytometer

Counting Cells Using Hemocytometer

Figure 4. The circle indicates the approximate area covered at 100× microscope magnification (10× ocular and 10× objective). Include cells on top and left touching middle line (O). Do not count cells touching middle line at bottom and right (Φ). Count 4 corner squares and middle square in both chambers (one chamber represented here).





T Mosmann
Journal of Immunological Methods 1983-12-16
A tetrazolium salt has been used to develop a quantitative colorimetric assay for mammalian cell survival and proliferation. The assay detects living, but not dead cells and the signal generated is dependent on the degree of activation of the cells. This method can therefore be used to measure cytotoxicity, proliferation or acti...Read More
F Denizot, R Lang
Journal of Immunological Methods 1986-05-22
A convenient way to estimate the number of viable cells growing in microtitre tray wells is to use a colorimetric assay and an automatic microplate scanning spectrophotometer. One such assay, developed by Mosmann, depends on the reduction by living cells of tetrazolium salt, MTT, to form a blue formazan product. However the orig...Read More
J Carmichael, W G DeGraff, A F Gazdar, J D Minna, J B Mitchell
Cancer Research 1987-02-15
Drug sensitivity assays were performed using a variation of a colorimetric [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT)] assay on V79, CHO-AuxB1, CHRC5, NCI-H460, and NCI-H249 cell lines following optimization of experimental conditions for each cell line. Results from this assay were compared with data as...Read More