In-Vitro Cytotoxicity

Assays
Cell viability assays assess how healthy the cells are by measuring markers of celular activity.
Cellviability determines how well or how poorly cells will respond to stress stimuli. 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 Assay is a homogeneous method to determine the number of viable cells in
culture. Cell viability and cytotoxicity assays are used for drug screening and
cytotoxicity tests
of chemicals.

VIABILITY testing is of high importance in many areas of cell

research including cytotoxicity
tests based on cell and tissue cultures, the
selection of proper tissue scaffolds for regenerative
medicine,quality assurance of products for transplantation research for
cancer treatment and
also for the inspection of hybridoma cells. For the evaluation of cell viability, flow cytometry
and microscopy are used besides the detection of cellular secretion products.

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

(a)
O(b) dead celI

live cells
 VIABILITY ASSAYS ARE BASED ON

measurement
Luminescence
ofmembrane
based
integrity
tests.

cellular
radioisot ope
respiration
incorporation

BASEDON MEMBRANEINTEGRITY

The damage to membrane may

occur due to cell disaggregation, cell
separation or and thawing. Membrane integrity can be
freezing
determined by uptakeof dyes to which viable
cells are impermeable (e.g.
naphthaleneblack, trypan blue, erythrosin)

Dye Dye Labelled

excusion Enzyme
uptake chromium release
Assay assav uptake assay Assavs

DYE EXCLUSION ASSAY

viable cells are imperneable to several dyes such as naphthalene black, trypan
blue, cosin Y,nigrosin grcenand eiythrocin B. (in plants- evan's blue stain)

The technique basically of mixing the cells in suspensionwith the dye

consists
•
observe under the microscopy.

Thestained cells and the total number of cells are counted.

The perccntage ofunstained cells represents the viable cells.

•The major limitation of this assay is that reproductively dead cells do not take up
the dye, and will be counted as though theyare viable.
 henslhingium dned
Dye Uptake Assay for Cytotoxicity(Naphthalene Black)

Principle:

The Dye Uptake Assay measures cytotoxicity by detecting the ability of

viable cells to
uptake and retain thedye Naphthalene Black. Dead or damaged cells fail to uptake the
dye.

Intact celi Fluorescein in

Pl and FOA s ndded intact cells

Advantages:

-Simpleand rapid method

-High sensitivity and specificity
- Non-toxic and non-radioactive dye

Limitations:

-May not detect cytostatic effects (inhibition of cellgrowth without cell death)
-Requires careful optimization of assay conditions

This assay is a useful tool for evaluating cytotoxicity in various research applications,
including pharmacology, toxicology, and biomedical research.

henctiagiam
Note: NaphthaleneBlack is a non-toxic, non-radioactive dye that is excluded by viable
cells but taken up by dead or damaged cells. The assay measures the ability of cells to

maintain membrane integrity and function.

 Dye Exclusion Assay
(Trypan Blue)for
Cytotoxicity
Principle:

The Dye Exclusion Assay

measures cytotoxicity by detecting the
exclude the dye
Trypan Blue. Dead ability of viable
remain unstained. or damaged cells cells to
take up the dye,
while viable cells

ml PBS
I
Irypan blug

Superst
Spin
Resuspcnde
Lelis

Cell Resuspend Mix 1:1 ratio of

pellct in PBS
sspension Incubatjon
Cell pellct
cell sample & (R.3tnin)
trypan bluc (4%
Stained cell W in PBS)

suspension

1LacmoCVioIcter Dead cells

-Live cells
Cell conting

Stained unstained
cells
Microscope abservcd under the
(B) microscope

Dead
celils
(stained blue)

Live ceils
(ustained)

Advantages:

-Simple and rapid method

- High
sensitivity and specificity
- Non-toxic and
non-radioactive dye

Limitations:

- Requiresmanual
counting or specialized
- May not detect cytostatic equipment
effects (inhibition of cell growth without cell death)
This assay is a widely used method for evaluating
cytotoxicity in various
applications, including research
pharmacology, toxicology, and
biomedical research.
 Note: Trypan Blue is a membrane-impermeabledye that is excluded by viable cells but
taken up by dead or damaged cells. The assay measuresthe ability of cells to maintain
membrane integrity and function.

Labelled Chromium Uptake Assay for Cytotoxicity

Principle:

The Labelled Chromium Uptake Assay measures cytotoxicity by detecting the release of
chromium-51 (51Cr) from labelled target cells. This assay is based on the principle that
viable cells retain chromium,while damaged or dead cells release it.

Advantages:

- Sensitive and quantitative measure of cytotoxicity

- Can detect both immune-mediatedand
chemical-induced cytotoxicity
- Relatively simple and fast assay

Limitations:

Requires radioactive material;s and specialized equipments

May not detect cytostatic defects (Inhibition of cell growth without celldeath)

This assay is widely used immunology;oncology and toxicology

in
research to evaluate
the cytotoxic effects of immune cells, drugs, and other agents.

Luminescence Test for Cytotoxicity

Principle:
The Luminescence Test measures cytotoxicity by detecting the reduction of cellular ATP
(adenosinetriphosphate) levels, which is indicative of cell death.
This assay uses a
luminescent substrate that reacts with ATP to produce light.
egsinevha
ATP
ATP ATP
ATP
ATP
Lyse cell
Firefly
luciferase
+ ATP + O,
Light
Mg
Luciferin
 Advantages:

-Fast and sensitive method for detectingcytotoxicity

- Can be used for high-throughput screening
-Non-radioactive and non-toxic reagents

Limitations:

- May not detect cytostatic effects (inhibition of cell growth without cell death)

- Requires specialized equipment(luminometer or plate reader)

This assay is widely used in pharmacology, toxicology, and biomedical research to

evaluate the cytotoxic efects of various compounds, including drugs, toxins, and
environmental pollutants.

Tetrazolium Reduction Assay (MTT Assay)

Principle:

The MTT Assay measures cell viability and cytotoxicity by detecting the reduction
of the
yellow tetrazolium salt (MTT)to a purple
formazan product by mitochondrial enzymes in
living cells.

yellow purple
MTT <formazan
mitochondrial
dehydrogenase

Advantages:

- Simple, rapid,and cost-effective method

- Can be used for high-throughput
screening
- Non-radioactive and non-toxic reagents

Limitations:
 - May not detect cytostatic effects (inhibition of cell growth without cell death)
Requires careful optimization of assay conditions

This assay is widely used in pharmacology,

toxicology, and biomedical research to
evaluate the cytotoxic effects of various
compounds,including drugs, toxins, and
environmental pollutants

LDH Assay for Cytotoxicity

Principle:

The LDH Assay measures cytotoxicity by detecting the release of Lactate

Dehydrogenase(LDH) enzyme from damaged or dead cells. LDH is a key enzyme in
cellular respiration,catalyzing the conversion of lactate to pyruvate.

Lactate INT
NAD+
LDH Diaphorase
LDH
NADH

LDH Pyruvate NTFomaz

Advantages:

-Simpleand cost-effective method

- Can detect both acute and chronic cytotoxicity
- Non-radioactive and non-toxic reagents

Limitations:

- May not detect cytostatic effects (inhibition of cell growth without cell death)
-Requires careful optimization of assay conditions
 This assay is widely
used in pharmacology,
toxicology, and biomedical
evaluate the cytotoxic research to
effects of various
compounds, including drugs,
environmental pollutants. toxins, and

Cellular Respiration
Connection:

LDH plays a critical role in

anaerobic glycolysis, a key step
measuring LDH release, this assay in cellular
respiration.By
compounds on
indirectly assessesthe impactof
cellular
respiration and metabolism. cytotoxic

Warburg Manometer
Assay for Cytotoxicity
Principle:

The Warburg
Manometer Assay
measures cytotoxicity by
respiration, specifically the rate of detecting changes in
oxygen consumption cellular
and carbondioxide
production.
Advantages:

- Directly
measures cellular
- Can detect both
respirationand
metabolic
acuteand chronic activity
- High cytotoxicity
sensitivity and specificity

Limitations:

-Complex and
labor-intensive setup
-Requires
specialized equipmentand expertise
- Limited
throughput capacity

This assay is a
classic method for
cancer research and assessing cytotoxicity,
toxicology studies. particularly in the context of

Connection to Cellular
Respiration:

The Warburg
Manometer Assay directly
carbon dioxide measures the rates of
production, which are oxygen consumption
key indicators of cellular and
changes in these rates,
the assay assesses respiration.By detecting
cellular metabolism and the impactof
cytotoxic compounds
energy production." onna
Q. State the Principleof MIT Assay. Draw a flow chart of the protocol.

The tetrazolium reduction assays measuresomeaspect of general metabolism or an

enzymatic activity asa marker of viable cells. All of these assays require incubation of a

reagent with a population of viable cells to convert a substrate to a coloured or fluorescent

product that can be detected with a plate reader. MTT which is positively charged and
readily penetrates iable eukaryotic cells.

Principle: Viable cells with active metabolism convert MTT into a purple colored formazan
product with an absorbance maximum near 570nm. When cells die, they lose the ability to

convert MTT into formazan, thus colour formation serves as a useful and convenient marker
of only the viable cells.

NADH NAD
Br

N-NH
-CH CHa
CH3 CH3
MTT Formnazan

Theformazanproduct of the MTT tetrazolium accumulates asan insoluble precipitate inside

cells as well as being deposited near the cell surface and in the culture medium. The
formazanmust be solubilized prior to recording absorbance readings. Various solubilization

methods include using: acidified isopropanol, DMSO, dimethylformamide, SDS.

METHODOLOGY

The MIT (3(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) tetrazolium

(MIT) substrateis prepared in aphysiologically balanced solution.

Added to cells in culture, usually at å final concentration of 0.2 -0.5mgml

Incubated for 1 to 4 hours

The quantity of fomazan (presumably directy proportional to the number of viable cll) is

measured by absorbance at 570 nm using a plate reading spectophotometer