Cytotoxicity assays measure the toxicity of compounds on cells. There are several methods to assess cytotoxicity, including dye exclusion tests using trypan blue or congo red that selectively stain dead cells, and fluorescent tests using propidium iodide or alamar blue. Colorimetric assays also exist such as MTT, SRB, and Hoechst dye assays that measure cellular components like proteins or DNA to indicate cell viability. Cytotoxicity is determined by quantifying the number of dead cells or decrease in living cells after exposure to potential cytotoxic compounds.

1. Methods of Cytotoxicity Study
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General Pharmacology
PC 520: Semester I
Assignment
Assigned by:-
Dr Santosh Kumar Guru Sir
NIPER HYDERABAD
Presented by:-
Nandita Pandey
PC/2022/210
Pharmacology &
Toxicology Dept
NIPER HYDERABAD

2. What are Cytotoxicity Assay
• Cytotoxicity is defined as any cytotoxic substance which can be a drug or any chemical
which is toxic to the cells and ultimately results in the cell death.
• Cytotoxicity Assays are the assays that gives the measure of the cytotoxic potential of the
cytotoxic compound and the ability to cause cell damage and cell death.
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3. Principle of Cytotoxicity Assay
 Membrane Integrity is the feature most often used to detect the cells cultured in vitro to assess
whether they are alive or dead.
 Cells that have lost membrane integrity and allow movement of otherwise non-permeable
molecules are classified as non-viable or dead.
 Detection of dead cells is accomplished by measuring movement of molecules either into or out of
the cells across membranes that have become leaky and cannot be repaired.
 Dyes are the markers used to determine cell viability and cytotoxicity.
 Dyes such as trypan blue and many fluorogenic DNA binding dyes result in selective staining of
the dead cells because they have lost their membrane integrity.
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4. Methods to Assess Cytotoxicity
• Dye Exclusion test:-
Trypan Blue
Congo Red
Erythrosine B
Fluorometric tests:-
Alamar Blue
Propidium iodide
CFDA-AM
GF-AFC
• Colorimetric tests:-
MTT
MTS
XTT
WST-1
WST-8
LDH
SRB
NRU
Crystal Violet
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It has been shown that nonviable cells which take up the
dye do not respire, glycolyse, or extend cellular processes.

5. Trypan Blue
 The selective staining of dead cells with trypan blue and microscopic examination on a hemocytometer to
determine the cell number and percent viability in a population of cells.
 The general concept is that trypan blue is excluded from live cells, but penetrates dead cells with a
damaged plasma membrane.
 The mechanism of selective staining of dead cells may actually involve impermeability of aggregates of
trypan blue into the viable cells thus staining only the damaged cells with disrupted plasma membrane.
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6. Fluorescent DNA Binding Dyes That Penetrate Dead Cells
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There are many fluorescent DNA binding dyes to select from which are
generally considered to be non-permeable to viable cells and can be used
for detection of the accumulation of dead cells in culture.
DNA dyes that readily pass through the intact cell membrane and stain the nucleus
of live cells should be avoided for consideration for measuring dead cells.
The cell-impermeant, amine-reactive dye only
binds to the surface of the live cell, resulting in
very dim fluorescence. The dye can penetrate
the cell membrane in dead cells and will bind
to internal proteins, resulting in very bright
fluorescence.

7. Congo Red: In-Vitro tests for Amyloids
The Congo red Amyloid Stain is a metachromatic anionic dye used to determine whether or
not tissue slices contain amyloid.
Congo red staining for amyloid, principle is based on hydrogen bridge bonds forming with the
substrate’s carbohydrate component. Congo red is an anionic dye that can deposit itself on
amyloid fibrils, causing them to show a distinct dichroism when exposed to polarised
light.
Under transmitted light, the Congo red-stained tissue appears orange-red; however, under
polarised light, the amyloid deposits emerge as vivid green double-refraction pictures against a
dark backdrop.
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8. Images of Amyloids stained with Congo Red Stain
Amyloids are the abnormal cell which is
permeable to the dye (Liver, spleen, Kidney)
Renal biopsy. Congo red stain under polarized light
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9. Erythrosine B: a Versatile Colorimetric and Fluorescent
Vital Dye for Bacteria
 Incubation of erythrosine B with bacteria specifically stains membrane-compromised dead cells.
Live and dead cells can then be quantified via bright-field microscopy or any instrument that quantitate
fluorescence.
 Erythrosine B is a cherry pink dye composed of a fluorescein salt (i.e. sensitive to light at certain
wavelengths).
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Fluorescent Erythrosine B is preferable to trypan blue as a vital
exclusion dye for mammalian cells in monolayer culture.

10. Propidium Iodide
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Propidium Iodide can only pass through disordered areas of membrane of
dead cells and intercalates with the DNA of the nuclei, emitting red
fluorescence light.
Propidium iodide (PI) is a fluorescent
intercalating agent that can be used to
stain cells and nucleic acids.
Propidium iodide is used as a DNA stain in flow
cytometry to evaluate cell viability or DNA content in
cell cycle analysis, or in microscopy to visualize the
nucleus and other DNA-containing organelles.
Fluorescence Microscopy images of
Propidium iodide (PI) stained cell seeded
scaffolds retrieved after 2 and 10 days.

11. Alamar Blue
The Alamar Blue is a cell viability assay reagent which contains the cell
permeable, non-toxic, and weakly fluorescent blue indicator dye resazurin.
It is useful for cytokine bioassays, cell viability assays, and in
vitro cytotoxicity determinations as well as cell growth monitoring
Resazurin is used as an oxidation-reduction
(REDOX) indicator that undergoes colorimetric
change in response to cellular metabolic
reduction.
The reduced form, resorufin, is pink and highly
fluorescent, and the intensity of fluorescence
produced is proportional to the number of
living cells respiring. AlamarBlue is a direct
indicator of cell health and it detects the level of
oxidation during respiration, quantitatively
measuring cell viability and cytotoxicity
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12. MTT Cell Cytotoxicity Assay
MTT assay is used to evaluate the cytotoxicity.
MTT assay evaluates the effect of a drug on the proliferative index of the cell population.
This is a colorimetric assay that measures the reduction of yellow 3-(4,5- dimethythiazol-2-yl)-2,5-
diphenyl tetrazolium bromide (MTT) by mitochondrial succinate dehydrogenase.
In MTT assay, cells in the exponential phase of
growth are exposed to a cytotoxic drug.
The MTT dye enters into the cells and then
passes into the mitochondria where it get
reduced to an insoluble, colored (dark
purple) formazan product.
After addition of the drug, the cells are
allowed to proliferate for two to three
population-doubling times (PDTs)
Since reduction of MTT can only
occur in metabolically active cells
the level of activity is a measure of
the viability of the cells.
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14. Sulforhodamine B Colorimetric Assay for Cytotoxicity Screening
The sulforhodamine B (SRB) assay is used for cell density determination, based on the
measurement of cellular protein content
Sulforhodamine B (SRB) Cytotoxicity Assay is a sensitive, reproducible and easy-to-use
assay based on the ability of SRB to bind to protein components of cells that have been
fixed to tissue culture plates.
As the binding of SRB is stoichiometric, the amount of dye extracted from stained cells is
directly proportional to the cell mass. The fixed dye is solubilized and is measured
photometrically at OD 540 nm with a reference filter of 690 nm. The OD values correlate
with total protein content and therefore with cell number.
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15. Hoechst Dye for DNA Binding
Hoechst dyes are cell- permeable and can bind to DNA in live or fixed cells.
Hoechst stains will selectively bind to A-T rich regions of double-stranded DNA, specifically binding to
the minor groove. Upon binding to DNA, Hoechst stains will undergo an approximately 30-fold increase in
fluorescence. The Hoechst stains can be excited by ultraviolet light (~360 nm) and will emit a blue
fluorescence (~460 nm).
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REFERNCES
1) https://youtu.be/qDWm6hqYFzM
2)https://youtu.be/HunKhtVukTE
3) https://www.ncbi.nlm.nih.gov/books/NBK540958/
4) https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4535150/

17. THANK YOU
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