Cell viability assays determine the number of living cells in a culture sample. There are several types of assays, including those measuring enzyme activity, membrane permeability, ATP production, DNA synthesis, and metabolic activity. Metabolic assays use tetrazolium salts or Alamar blue that change color when reduced by living cells, allowing quantification with a spectrophotometer. Membrane integrity assays use dyes like propidium iodide to distinguish living from dead cells based on intact versus compromised membranes.

1. Cell Viability Assays
Department of Pharmacology

2. Viability assays
• Cell Viability Assay is a homogeneous method to determine the
number of viable cells in culture.
• Cell viability, defined as the number of healthy cells in a sample,
determines the amount of cells (regardless of phase around the cell
cycle) that are living or dead, based on a total cell sample.
• Cell viability and cytotoxicity assays are used for drug screening
and cytotoxicity tests of chemicals.

3. They are based on various cell functions such as
• Enzyme activity,
• Cell membrane permeability,
• Cell adherence, ATP production,
• Co-enzyme production
• Nucleotide uptake activity

4. DNA synthesis cell proliferation assays
• One of the most reliable and accurate assay types is measurement
of DNA synthesized in the presence of a label.
• Traditional cell proliferation assays involve incubating cells for a
few hours to overnight with 3H-thymidine.
• Proliferating cells incorporate the radioactive label into their
nascent DNA, which can be washed, adhered to filters and then
measured using a scintillation counter.

5. Metabolic cell proliferation assays
• Another measure of cell proliferation is the metabolic activity of a
population of cells.
• Tetrazolium salts or Alamar Blue are compounds that become
reduced in the environment of metabolically active cells, forming
a formazan dye that subsequently changes the color of the media.
• This is caused by increased activity of the enzyme lactate
dehydrogenase during proliferation.
• The absorption of the media-containing dye solution can be read
using a spectrophotometer or microplate reader in low- or high-
throughput configurations.

6. • Four types of tetrazolium salts are most common
 MTT
 XTT
 MTS
 WST1
• A disadvantage of MTT is that it is insoluble in standard culture
medium, and the formazan crystals produced during reduction must
be dissolved in DMSO or isopropanol.
• Because of this, MTT is mainly an endpoint assay.
• The other salts, as well as Alamar Blue, are soluble in culture media
and are nontoxic.
• They can be used for continuous monitoring, to follow dynamic
changes in proliferation over time.

7. • XTT reduces less efficiently and may need additional factors added.
• WST1 is more sensitive, reduces more efficiently and shows faster
color development compared to the other salts.
• Alamar Blue is also sensitive, capable of detecting as few as 100 cells
in a well of a microtiter plate.
• The tetrazolium salts and Alamar Blue redox dyes can be quantified
with a range of instruments for conventional or high-throughput
studies
• For example, standard spectrophotometers or spectrofluorometers or
plate readers for spectrophotometric or spectrofluorometric microtiter
well plates.

8. Tetrazolium Reduction Assays
• The most commonly used compounds include: MTT, MTS, XTT, and
WST-1.
• These compounds fall into two basic categories:
 MTT which is positively charged and readily penetrates
viable eukaryotic cells and
 Those such as MTS, XTT, and WST-1 which are negatively
charged and do not readily penetrate cells.
• The latter class (MTS, XTT, WST-1) are typically used with an
intermediate electron acceptor that can transfer electrons from the
cytoplasm or plasma membrane to facilitate the reduction of the
tetrazolium into the colored formazan product.

9. MTT Tetrazolium Assay Concept
• This is a colorimetric assay that measures the reduction of yellow 3-
(4,5- dimethythiazol2-yl)-2,5-diphenyl tetrazolium bromide (MTT)
by mitochondrial succinate dehydrogenase.
• The MTT enters the cells and passes into the mitochondria where it
is reduced to an insoluble, coloured (dark purple) formazan
product.
• The cells are then solubilised with an organic solvent (eg.
isopropanol) and the released, solubilised formazan reagent is
measured spectrophotometrically.
• Since reduction of MTT can only occur in metabolically active cells
the level of activity is a measure of the viability of the cells

11. Measuring ATP
• It takes advantage of the tight regulation of intracellular ATP within
cells.
• Dying or dead cells contain little to no ATP, so there is a tight linear
relationship between cell number and the concentration of ATP
measured in a cell lysate or extract.
• The bioluminescence-based detection of ATP, using the enzyme
luciferase and its substrate luciferin, provides a very sensitive readout.
• In the presence of ATP, luciferase produces light (proportional to the
ATP concentration) that can be detected by a luminometer or any
microplate reader capable of reading luminescent signals.
• This approach is also well suited to high-throughput cell proliferation
assays and screening.

12. • ATP has been widely accepted as a valid marker of viable cells.
• When cells lose membrane integrity, they lose the ability to
synthesize ATP and endogenous ATPases rapidly deplete any
remaining ATP from the cytoplasm.
• The ATP detection reagent contains detergent to lyse the cells,
ATPase inhibitors to stabilize the ATP that is released from the
lysed cells, luciferin as a substrate, and the stable form of luciferase
to catalyze the reaction that generates photons of light.

14. Sulforhodamine B Cell Cytotoxicity Assay
• This assay relies on the ability of SRB to bind cellular protein
components and measure the total biomass.
• SRB is a bright-pink aminoxanthene dye that can form an electrostatic
complex with basic amino acid residues of proteins in slightly acidic
conditions but it can dissociate under basic conditions.
• It has been widely used for drug toxicity screening against different
types of cancerous and non-cancerous cell lines.
• In addition, this assay is independent of cell metabolic activity and
therefore should show less interference by the testing compounds.
• Since the binding of SRB is stoichiometric, the incorporated dye
released from stained cells after washing is directly proportional to the
cell biomass and can be measured at 565 nm

17. Plasma membrane integrity
• Assessing cell membrane integrity is one of the most common and
straightforward ways to measure cell viability and assess
cytotoxic consequences.
• Compounds that have cytotoxic effects often compromise cell
membrane integrity and induce necrosis.
• Dyes, such as propidium iodide and 7-AAD, are normally
excluded from the inside of healthy cells; however, if the cell
membrane has been compromised, they freely cross the
membrane and stain intracellular components.
• This method distinguishes healthy cells with uncompromised
membrane integrity (unlabeled) from non-healthy ones (colored).