The document discusses the measurement of cell viability and cytotoxicity, defining both terms and outlining various assessment methods including dye exclusion, colorimetric, enzyme release, fluorometric, and luminometric assays. It explains how these methods work, their principles, and specific examples such as the MTT and XTT assays for colorimetric analysis, and the ATP assay for luminometric analysis. Additionally, it provides practice questions for learning about the principles and applications of these assays.

1. Animal cell science & Technology
3. Cell cytotoxicity
Shailendra Singh Shera, Ph.D

2. Measurement of viability and cytotoxicity
Outline
1. Definition of Viability & Cytotoxicity
2. Methods of assessment
a) Dye Exclusion methods
b) Colorimetric Assay
c) Enzyme release assay
d) Fluorometric assay
e) Luminometric assay
3. Practice Questions

3. Definition: Viability and Cytotoxicity
Cells in culture should be routinely checked for their health status to ascertain their normal
physiological growth. There are various methods to ascertain the health status of growing cells.
These are based on various cell functions such as enzyme activity, cell membrane permeability, cell
adherence, ATP production, co-enzyme production, and nucleotide uptake activity. Before going
further, let’s start with definition:
What is viability?
Viability of the cells represents the capability of their existence, survival and developments.
What is cytotoxicity?
Chemicals with a capability to destroy cells i.e toxic/ harmful to cells causing either cells death or
deviation from normal physiological behavior.

4. Methods of Assessment
Cell viability and cytotoxicity assays are based on colorimetric, fluorometric and bioluminescent
detection chemistries. The tree below shows various modes of assessing viability and
cytotoxicity.
Cell viability and
cytotoxicty
Dye exclusion
method
Colorimetric
assay
Fluorometric
assay
Luminometric
assay

5. Dye exclusion methods
This techniques is based on exclusion of dye by viable cells.
Dead cells have damaged plasma membrane and increased permeability to dyes. Therefore, Viable cells remains
unstained whereas dead cells are stained.
Common dyes:
Trypan blue: It selectively stains dead cells. Staining with trypan blue followed by microscopic examination on
hemocytometer is frequently used method to determine the cell number and percent viability in a population of
cells.
Dead cells are stained blue
Erythrosine B : Erythrosine B, also known as erythrosine or Red No. 3 is a vital stain.
It also has fluorescent capacity.
The principle is similar to Trypan Blue. It asses memrane integrity and penetrates plasma membrane of dead
cells.
Fluorescein diacetate (FDA): Fluorescein diacetate (FDA) is a non-fluorescent molecule, which is hydrolysed to
fluorescent fluorescein in live cells.
It is also a vital stains.
The hydrolysis is performed by esterase enzymes present in viable cells only.
viable cells retain the dye and fluoresce whereas nonviable cells do not. Fluorescein diacetate is not cytotoxic.

6. Colorimetric assay
Colorimetric assays: MTT assay, MTS assay, XTT assay, WST-1 assay, WST-8 assay, LDH assay, SRB assay,
NRU assay and crystal violet assay.
Principle:
•It is the measurement of a biochemical marker to evaluate metabolic activity of the cells.
•Reagents used in colorimetric assays develop a color in response to the viability of cells, allowing the colorimetric
measurement of cell viability via spectrophotometer
MTT assay
The MTT assay is colorimetric assay which uses reduction of a yellow tetrazolium salt (3-(4,5-dimethylthiazol-2-
yl)-2,5-diphenyltetrazolium bromide, or MTT.
It is based on the ability of mitochondrial nicotinamide adenine dinucleotide phosphate (NADPH)-dependent
cellular oxidoreductase enzymes to reduce the tetrazolium dye MTT to its insoluble formazan, which has a purple
color.
Formazan crystals are then dissolved using a solubilizing solution and absorbance is measured at 500-600
nanometers using a plate-reader.
The darker the solution, the greater the number of viable, metabolically active cells.
During spectrophotometric determination, the absorbance value will increase with time. % viability is calcultaed
as:

7. XTT Assay
 It is based on the reduction of a yellow tetrazolium salt (sodium 3´-[1- (phenylaminocarbonyl)- 3,4-
tetrazolium]-bis (4-methoxy6-nitro) benzene sulfonic acid hydrate or XTT) to an orange formazan
dye by metabolically active cells
XTT is simply for measuring proliferation and is therefore an excellent solution for quantitating cells
and determining their viability.
The XTT assay is used to measure cellular metabolic activity as an indicator of cell viability,
proliferation and cytotoxicity.
It is used for the measurement of cell proliferation in response to growth factors, cytokines and
nutrients
https://www.sigmaaldrich.com/technical-documents/protocols/biology/roche/cell-proliferation-kit-xtt-assay.html

8. Enzyme release assays:
The membrane integrity of cells can also be assessed by estimating the enzymes released.
Lactate dehydrogenase (LDH) has been the most widely used enzyme for this purpose.
The damaged / non viable cells with compromised membrane integrity releases lactate
dehydrogenase. LDH converts Lactate to pyruvate, generating NADH from NAD. The NADH
interacts with the probe to generate color at 450 nm.
The amount of colored product formed is directly proportional to the LDH activity in the sample.
The LDH activity is determined as NADH oxidation or INT reduction over a defined time period.
The resulting red formazan absorbs maximally at 492 nm and can be measured quantitatively at
490 nm.

9. Fluorometric assay
Fluorometric assays: alamarBlue assay and CFDA-AM assay.
Fluorometric assays of cell viability and cytotoxicity are easy to perform with the use of a
fluorescence microscope, fluorometer, fluorescence microplate reader or flow cytometer.
Fluorometric assays are also applicable for adherent or suspended cell lines and easy to use.
Alamar Blue
AlamarBlue Cell Viability Reagent is an indigo-colored, non-toxic reagent that detects
metabolically active cells and is used for the quantitative analysis of cell viability and proliferation
Resazurin, the active ingredient of alamar Blue reagent upon uptake by living cells is reduced to
resorufin, a compound that is red in color and highly fluorescent.
Absorbance- or fluorescence-based plate reader are used to assess change in viability.
Absorbance (detected at 570 and 600 nm) or fluorescence (using an excitation between 530–
560 and an emission at 590 nm).
Add alamar blue
solution to complete
media directly
Incubate
1-4 hours
Spectrophotometric/
Fluorometric
observation

10. Luminometric assay
Luminometric assays: ATP assay and real-time viability assay.
ATP assay
•When cells damaged lethally and lose membrane integrity, they lose the ability to synthetize
ATP and the ATP level of cells decreases dramatically.
•The ATP assay is based on the reaction of luciferin to oxyluciferin. Enzyme luciferase catalyzes
this reaction in the presence of Mg2+ ions and ATP yielding a luminescent signal.
•There is a linear relationship between the intensity of luminescent signal and ATP concentration
or cell number

11. 1. MTT assay is a type of
a. Colorimetric assay
b. Fluorometric assay
c. Emzyme release assay
d. None of the above
2. What is the property of vital stain?
a. It should kill cells in culture on addition
b. It should be non toxic to cells in culture
c. Both (a) and (b)
d. None of the above
3. Dye exclusion method assays
a. Membrane integrity of cell
b. Enzyme reduction capability.
c. Integrity of DNA
d. None of the above
4. ATP assay is an example of
a. Luminometric method
b. Colorimetric method
c. Enzyme release
d. DNA binding
5. Fluorescein diacetate (FDA) is hydrolyzed by cellular esterase to …………
a. Resazurin
b. Fluorescein
c. Formazan
d. Dimethyl sulphoxide
*Practice and Learn Animal cell Science and Technology: Multiple choice question for learning.
Author: Shailendra Singh Shera . Publisher: Amazon Kindle.
Practice questions: MCQs*

12. 1. https://www.sigmaaldrich.com/technical-documents/protocols/biology/roche/cell-proliferation-kit-
xtt-assay.html
2. https://www.thermofisher.com/order/catalog/product/DAL1025#/DAL1025
3. Aslanturk O.S.“In-vitro-cytotoxicity-and-cell-viability-assays-principles-advantages-and-
disadvantages.” Intechopen. DOI: 10.5772/intechopen.71923
Practice MCQs
1. Practice and Learn Animal cell Science and Technology: Multiple choice question for learning.
Author: Shailendra Singh Shera . Publisher: Amazon Kindle.
References & Further reading