This document discusses cytotoxicity and methods for measuring it in vitro. It defines cytotoxicity as the ability of chemicals or cells to destroy living cells, which can lead to necrosis, apoptosis, or cytostasis. Measuring cytotoxicity is important for drug development and safety testing. In vitro assays are now commonly used as they are faster, cheaper, and more accurate than animal models. Several types of cytotoxicity assays are described, including dye exclusion assays like trypan blue, colorimetric assays like MTT, fluorometric assays like CFDA-AM, and luminometric assays like ATP assays. These assays measure endpoints such as membrane integrity, enzyme activity, proliferation, and ATP production to determine cytotoxic effects of chemicals on cells.

1. CYTOTOXICITY

2. Contents
■ Introduction
■ Importance of measuring cytotoxicity
■ in vitro cytotoxicity studies
■ How to measure cytotoxicity
■ Cytotoxicity assays
I. Dye exclusion
II. Fluorometric
III. Colorimetric
IV. luminometric

3. Introduction
Cytotoxicity
■ The ability of certain chemicals or mediator cells to destroy living cells.
■ can be caused by
1. chemical stimuli (drugs, cosmetics etc.)
2. exposure to other cells (NK andT cells)
3. physical/environmental conditions (radiation exposure, temperature or
pressure extremes, etc.)

4. Cytotoxicity can lead healthy living cells to three potential cellular fates
1. Necrosis (accidental cell death)
■ Rapid loss of membrane integrity and cell lysis
2. Apoptosis (programmed cell death)
■ slower, more orderly, and genetically controlled
3. Cytostasis (a decrease in cell viability)
■ cells remain alive but fail to actively grow and divide

5. Importance of measuring cytotoxicity
Two major reasons
1. either you want specific cells to die and look for an adequate
compound/condition (cancer and immunotherapy)
2. or you want to exclude cytotoxicity in specific cells (chemicals and
drugs)
Applications
■ Drug discovery process
■ oncological research
■ Safety evaluation of pesticides, plant extracts, food additives, cosmetics
and industrial chemicals

6. Invitro cytotoxicity studies
Before the advent of tissue culture technology whole model based studies were conducted to
measure cytotoxicity i.e. using whole plants or animal models.
■ It was a tedious process, having low prediction probability and mostly resulted in death of
model animals.
■ Now tissue culture technology have made it possible to measure cytotoxicity on specific
cultured cell lines giving more accurate results.
Advantages of In vitro cytotoxicity and/or cell viability
■ speed
■ reduced cost
■ potential for automation
■ More prediction probability (testing on specific cell lines)
■ Do not require testing models i.e. plants and animals
■ Useful for testing large number of samples

7. How to measure cytotoxicity
■ It is assumed that a viable cell will express the following signs of livelihood and functionality:
i. It will metabolize
ii. It will divide and proliferate
iii. It will have intact membrane integrity
Based on these assumptions various assays have been developed which measure cytotoxicity
and cell viability by certain cellular functions such as
■ cell membrane permeability,
■ enzyme activity,
■ cell adherence,
■ ATP production,
■ co-enzyme production,
■ nucleotide uptake activity

8. Cytotoxicity assays
In order to determine the cell death caused by various mechanisms, there is a need
for cytotoxicity and cell viability assays which are,
■ cheap
■ reliable
■ reproducible
These assays can be classified according to measurement types of end points (color
changes, fluorescence, luminescent etc.)
I. Dye exclusion: Trypan blue, eosin, Congo red, erythrosine B assays.
II. Colorimetric assays: MTT assay, MTS assay, XTT assay,WST-1 assay,WST-8
assay, LDH assay, SRB assay, NRU assay and crystal violet assay.
III. Fluorometric assays: alamarBlue assay and CFDA-AM assay.
IV. Luminometric assays: ATP assay and real-time viability assay

9. Dye exclusion assays
■ viable cells exclude dyes, but dead cells not exclude them.
■ Determination of membrane integrity is possible
■ A variety of such dyes have been employed, including eosin, Congo red,
erythrosine B, and trypan blue.
I. comparatively simple
II. require small numbers of cells
III. rapid,
IV. capable of detecting cell kill in nondividing cell populations
■ these dyes are not recommended for use on monolayer cell cultures but rather
they are intended for cells in suspension.

10. Dye exclusion assays
Trypan blue assay
■ live cells possess intact cell membranes that
exclude this dye, whereas dead cells do not.
■ Cell suspension is mixed with dye and then
visually examined.
■ Viable cells will have a clear cytoplasm, whereas
dead cells will have a blue cytoplasm.
■ counting is generally done using a
hemocytometer.

11. Colorimetric assays
■ Reagents used in colorimetric assays develop a color in response to the viability of cells.
they evaluate metabolic activity of cells
■ applicable for adherent or suspended cell lines
I. easy to perform
II. comparably economical
MTT assay (MTT (3-(4,5-dimethylthiazol-2-yl)-2–5-diphenyltetrazolium bromide)
■ It measures cell viability through determination of mitochondrial function of cells by
measuring activity of mitochondrial enzymes such as succinate dehydrogenase.
■ MTT or close dyes (MTS, XTT, WSTs) are reduced to a purple formazan by NADH.
■ This product can be quantified by light absorbance at a specific wavelength.

13. Colorimetric assays
LDH (lactate dehydrogenase) assay
■ Measures the stable, cytosolic, lactate dehydrogenase (LDH) enzyme
quantitatively.
■ LDH is an enzyme that is normally found within the cell cytoplasm.
■ Due to loss of membrane integrity LDH enzyme is released into the cell culture
medium from damaged cells.
■ The released LDH is measured with a coupled enzymatic reaction that results in
the conversion of a tetrazolium salt (iodonitrotetrazolium (INT)) into a red color
formazan by diaphorase.

14. Fig: LDH assay

15. Fluorometric assays
■ easy to perform with the use of a fluorescence microscope, fluorometer,
fluorescence microplate reader or flow cytometer.
■ applicable for adherent or suspended cell lines
■ easy to use
■ more sensitive than colorimetric assays
CFDA-AM assay
■ CFDA-AM (5-carboxyfluorescein diacetate, acetoxymethyl ester) is a fluorogenic
dye that is used for cytotoxicity determination.
■ The conversion of CFDA-AM to fluorescent dye, CF (carboxyfluorescein) by the
estarases in cells indicates the integrity of plasma membrane.
■ Viable cell will show green fluorescence.

16. Fig: CFDA-AM assay and flowcytometric
results

17. Luminometric assays
■ fast and simple determination of cell proliferation and cytotoxicity in mammalian
cells.
■ produces persistent and stable glow-type signal after reagent addition.
ATP assay
■ When cells are damaged lethally, 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.
■ fastest cell viability assay to use, the most sensitive, and is less prone to artifacts

18. Fig: ATP assay

19. Conclusion
■ A broad spectrum of cytotoxicity and cell viability assays are currently
used in the fields of toxicology and pharmacology.
■ Tissue culturing techniques have solved many problems associated with
conventional cytotoxicity assays.
■ If it is possible, more than one assays should be used to determine
cytotoxicity and/or cell viability in in vitro studies.
■ Thus, reliability of the obtained results would increase.

20. Thank you!