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Cell viability Assays
• Cell Viability Assay is a homogeneous method
to determine the number of viable cells in
• 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
They are based on various cell functions such as
• enzyme activity,
• cell membrane permeability,
• cell adherence, ATP production,
• co-enzyme production, and
• nucleotide uptake activity
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
• Proliferating cells incorporate the radioactive
label into their nascent DNA, which can be
washed, adhered to filters and then measured
using a scintillation counter.
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
• The absorption of the media-containing dye solution can be read using a
spectrophotometer or microplate reader in low- or high-throughput
• Four types of tetrazolium salts are most common:MTT,XTT MTS and 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.
• 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 using,
for example, standard spectrophotometers or spectrofluorometers or
plate readers for spectrophotometric or spectrofluorometric microtiter
Tetrazolium Reduction Assays
• The most commonly used compounds include: MTT, MTS, XTT, and WST-1.
These compounds fall into two basic categories:
• 1) MTT which is positively charged and readily penetrates viable
eukaryotic cells and
• 2) 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.
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
• Since reduction of MTT can only occur in metabolically active cells the
level of activity is a measure of the viability of the cells
• It takes advantage of the tight regulation of intracellular ATP within
• 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
• 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.
• 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
• 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.
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
• 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
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
• This method distinguishes healthy cells with uncompromised membrane
integrity (unlabeled) from non-healthy ones (colored).