A Guide to Assaying Cell Viability

Most cell viability assays depend on measuring cell metabolism and enzyme activity. These are methods that correspond to the number of living cells within the population.

Methods of Cell Viability Assay

  • Dyes that are reduced by enzymes in the cell
  • Dyes that depend upon the mitochondrial membrane potential
  • Dyes cleaved by cellular esterase
  • Assays for ATP and ADP
  • Assays of glycolytic flux and oxygen consumption

The most commonly used Abcam assays for cell viability are those based on MTS, resazurin, TMRE, calcein violet, and ATP luminescence.

There are other indirect cell viability assays that depend upon the measurement of cytotoxicity. This parameter refers to the number of cells in a population that are dead or damaged. Some methods of doing this are the LDH assay or with dyes like 7-AAD to measure damage to cell membranes.

At times the assay used measures apoptosis, such as that involving Annexin V, TUNEL or caspase assays. Another is the cell proliferation or cell cycle assay, like those which use dye dilution, BrdU/EdU, or DNA staining dyes.

Dye Reduction Assays

The tetrazolium assays for cell viability depend on the action of cell dehydrogenases to form a formazan product which is colored. The concentration of this product is measured using the absorbance property. Other assays use resazurin reduction, which occurs when electrons are accepted from the mitochondrial respiratory chain, and which results in the formation of resorufin, a fluorescent product.

Tetrazolium Assay Family

The following table shows the tetrazolium assay family:

Assay Instrument Notes Assay kits
MTT Plate reader Original tetrazolium assay; still very popular. Only tetrazolium assay that needs a wash/solubilization step. ab211091
MTS Most popular assay. More heavily used than WST-1. ab197010
WST-1 More sensitive than MTT, XTT or MTS. ab155902
Cell Counting Kit-8/CCK-8/ WST-8 ab228554
XTT assay ab232856

Resazurin Family

Resazurin is equivalent to the active ingredient of ThermoFisher’s alamarBlue®

Assay Instrument Notes Assay kits
Resazurin Plate reader, microscope, flow cytometer Fluorometric (Ex/Em 535–560/560–615) or colorimetric. No-wash assay. Fluorescent
readout enables multiplexing with other assays.

Dyes Dependent on the Mitochondrial Membrane Potential

Many dyes are available that build up within the mitochondria because of the membrane potential of these organelles. They identify viable cells. When membrane potential is lost, staining disappears, and this indicates apoptosis. The table below shows the assays in this group:

Assay Instrument Notes Assay kits
TMRE/TMRM Plate reader, microscope, flow cytometer Most popular Abcam mitochondrial membrane
dye assay. Ex/Em 549/575 nm. Washed out of mitochondria after fixation.
JC-1/JC-10 JC-1 (Ex/Em 530/530–570) and JC-10 (Ex/Em 590/520–570) form red aggregates at high concentrations (unaggregated dye is green). Loss
of membrane potential causes loss of dye and increased green fluorescence. JC-10 is more
soluble than JC-1. Washed out after fixation.
JC-1: ab113850
Mitotracker Red Ex/Em 579 /599. Not washed out after fixation.
Rhodamine 123 Ex/Em 507/529. Washed out after fixation.
MitoNIR Plate reader, flow cytometer Ex/Em 635/660. ab112149
MitoOrange Ex/Em 540/590. ab138898

Esterase Cleavage

Dyes which are hydrophobic, like calcein, enter cells by diffusion and are cleaved by esterases inside the live cells to form hydrophilic fluorescent compounds, that remain intracellular. The following table shows these assays:

Assay Instrument Notes Assay kits
Calcein AM Plate reader, microscope,
flow cytometer
Ex/Em 495/515 nm ab228556
Calcein violet AM Plate reader, microscope,
flow cytometer
Ex/Em 405/460 nm ab176748
Esterase-cleaved blue Plate reader Ex/Em 360/450 nm ab112120
Esterase-cleaved green Plate reader, microscope Ex/Em 490/520 nm ab112122
Esterase-cleaved near IR Plate reader Ex/Em 633/660 nm ab112123

ATP Assays

Most of the assays in this group rely on a compound which causes cell membrane permeabilization, resulting in the release of ATP, which leads to light production via ATP-dependent luciferase. Some use ATP-dependent glycerol phosphorylation or the phosphorylation of another substrate. Some of the assays of this type are shown in the table below:

Assay Instrument Notes Assay kits
Luminescence ATP assay Luminometric plate reader No-wash assay. ab113849
Luminescence ADP/ATP assay No-wash assay. After ATP analysis, ADP is converted to ATP for detection. ab65313
ATP phosphorylation assay Plate reader No-wash assay used with cell lysates. Not
as sensitive as luminescence assays. Fluorometric (Ex/Em 535/587 nm) is more sensitive than colorimetric.

Assays Dependent on the Consumption of Oxygen or the Rate of Glycolysis

The oxygen consumption rate shows how metabolically active the cell is. Examining the levels of oxygen and the glycolytic flux inside the cell gives greater detail about the cellular metabolism. The relevant assays are shown below:

Assay Instrument Notes Assay kits
Extracellular oxygen consumption Plate reader No-wash assay. Dye signal (Ex/Em 380/650 nm) increases as respiration lowers O2 levels. No need for specialized instruments. ab197243
Intracellular oxygen levels Dye fluorescence (Ex/Em 340/642) is quenched by intracellular oxygen. No-wash assay. ab197245
Glycolysis activity No-wash assay. Lactate production causes extracellular acidification and increased dye fluorescence (Ex/Em 340-380/615 nm). ab197244

About Abcam

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Last updated: Apr 1, 2019 at 6:17 AM


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