This document discusses various assays for measuring cell viability and proliferation, including metabolic assays that measure enzymatic activity (MTT, XTT, WST-1), fluorescent assays using dyes (CFSE, live/dead staining, Hoechst 33342), and trypan blue staining. It provides details on how each assay works and what cellular parameters it measures, such as viable cell number, proliferation rate, or cytotoxic effects of compounds.

1. CELL VIABILITY AND
PROLIFERATION ASSAYS
Presented by:-
Mohit Agrawal
M.Pharm (Pharmacology)
Delhi Pharmaceutical Sciences and Research University, New
Delhi

2. INTRODUCTION
 Cell viability assay is a homogeneous method to determine the
no. of viable cell in culture.
 Cell viability assay method is used to estimate the number of
viable cells in multi-well plates.
 Cell-based assays are often used for screening 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.

3. DNA SYNTHESIS PROLIFERATION ASSAYS
 BrdU Cell Proliferation Assay
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 assay) may be used
instead of thymidine. Cells that have incorporated BrdU into their DNA
are easily detected using a monoclonal antibody against BrdU and an
enzyme- or fluorochrome-conjugated secondary antibody.

4.  EdU Proliferation Assays
Base click 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.

5. 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, and WST-1 to
colored formazan compounds or the bio-reduction of resazurin
occurs only in metabolically active cells.
Actively proliferating cells increase their metabolic activity, while
cells exposed to toxins will have decreased activity.

6. 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 using absorbance as a function of
concentration of converted dye.

7. XTT CELL PROLIFERATION ASSAYS
In contrast to MTT, the cleavage product of XTT is soluble in water.
The tetrazolium salt XTT is cleaved to formazan by a complex
cellular mechanism.
This bio-reduction occurs in viable cells only, and is related to
NAD(P)H production through glycolysis.
The amount of formazan dye formed directly correlates to the
number of metabolically active cells in the culture.

8. 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 bio-reduction is largely dependent on the glycolytic production
of NAD(P)H in viable cells.
The amount of formazan dye formed directly correlates to the
number of metabolically active cells in the culture.

9. LUMINISCENT CELL PROLIFERATION ASSAYS
Because ATP is an indicator of metabolically active cells, the number
of viable cells can be assessed based on the amount of ATP available.
The ATP viability cell luciferase offers a highly sensitive homogenous
assay for quantifying ATP in cell cultures.
This kit employs firefly luciferase to oxidize D-Luciferin and the
resulting production of light to assess the amount of ATP available in
cell cultures.
By relating the amount of ATP to the number of viable cells, the assay
has wide applications, ranging from the determination of viable cell
numbers to cell proliferation to cell cytotoxicity.

10. FLOURESCENT DYE PROLIFERATION ASSAY
 CFSE Labeling
5(6)-Carboxyfluorescein diacetate N-succinimidyl ester (CFSE) is a
popular choice for measuring the number of divisions undergone by
a cellular population.
Upon entering the cell, CFSE is cleaved by intracellular esterase 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 enabling simple detection of the number of cell divisions by
flow cytometry.

11.  Live/Dead Cell Double Staining
Live/Dead Cell Double Staining can be utilized for simultaneous
fluorescence detection 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). Calcein-AM therefore only stains viable cells.
In contrast, 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 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 single-
excitation fluorescence microscope.

12. 3D CELL CULTURE LIVE/DEAD/TOTAL CELL
TRIPLE STRAINING
The Cell Viability Imaging Kit is a three-color assay that can be used with 2D and
3D cell cultures for simultaneous fluorescence staining of viable cells (Calcein-AM),
dead cells (Propidium Iodide/PI), as well as total cells (Hoechst 33342).
Calcein-AM fluoresces green on binding calcium, relying on esterase activity
present only in metabolically-active viable cells.
Propidium Iodide (PI) is a nuclear dye that is excluded by the membrane of live
cells, but passes through the damaged membrane of dead cells, intercalating with
the DNA to emit a strong red fluorescence.
Hoechst 33342 is a DNA staining dye that exhibits low cytotoxicity. It fluoresces
blue and is used as an indicator of total cell count.

13. TRYPAN BLUE CELL COUNTING
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 the blue dye, whereas dead (non-viable) cells do.
Cell viability can be calculated using the ratio of total live/total cells
(live and dead). Staining also facilitates the visualization of overall cell
morphology.
Trypan Blue has a greater affinity for serum proteins than for cellular
protein.