Assay

1. Submitted By
Dr. Neha Sharma
Guest Faculty
SOS Biotechnology
Subject: Animal Biotechnology (BT-203)

2. Cell viability assays assess how healthy the cells are by measuring
markers of cellular.
activity. Cell viability 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 assays

3. 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

4. HOW??
• Example:
• Live cell count: 2,337,500 cells/mL
• Dead cell count: 50,000 cells/mL
• 2,337,500 + 50,000 = 2,387,500 cells
• 2,337,500 ÷2,387,500 = 97.9% viability

5. VIABILITYASSAYS ARE BASED ON
measurement
of membrane
integrity
cellular
respiration
radioisotope
incorporation
Luminescence
based
tests.

6. BASED ON MEMBRANE INTEGRITY
The damage to membrane may occur due to cell disaggregation, cell
separation or freezing and thawing. Membrane integrity can be
determined by uptake of dyes to which viable cells are impermeable (e.g.
naphthalene black, trypan blue, erythrosin)
Dye
exclusion
Assay
Dye
uptake
assay
Labelled
chromium
uptake assay
Enzyme
release
Assays

7. DYE EXCLUSION ASSAY
• viable cells are impermeable to several dyes such as naphthalene black, trypan
blue, eosin Y, nigrosin green and erythrocin B. (in plants- evan’s blue stain)
• The technique basically consists of mixing the cells in suspension with the dye
observe under the microscopy.
The stained cells and the total number of cells are counted.
The percentage of unstained 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 they are viable.

8. DYE UPTAKE ASSAY
• The viable cells can take up the dye diacetyl fluorescein and hydrolyse it to
fluorescein. The latter is held up by the viable cells, as it is impermeable to
membrane. The viable cells therefore emit fluorescein green while the dead cells do
not. Thus, the viable cells can be identified.

9. LABELLED CHROMIUM UPTAKE ASSAY
• Labelled chromium (51Cr) binds to the intracellular proteins through basic amino
acids.
• When the cell membrane is damaged the labelled proteins leak out of the cell and
the degree of leakage is proportional to the amount of damage.

10. ENZYME RELEASE ASSAY
• 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.
• Principle: When using an LDH colorimetric assay, the amount of LDH released
in the surrounding environment is measured with an enzymatic reaction which
converts iodonitrotetrazolium or INT (a tetrazolium salt) into a red colour
formazan. When the cell membrane is damaged lactate dehydrogenase (LDH), is
released into the surrounding extracellular space. Since this only happens when cell
membrane integrity is compromised, the presence of this enzyme in the culture
medium can be used as a cell death marker.
• When LDH is present in the cell culture, it reduces NAD+ to NADH and H+ the
catalyst (diaphorase) then transfers H/H+ from NADH + H+ to the tetrazolium salt
INT forms the red coloured formazan salt. The amount of colour produced is
measured at 490nm and is proportional to the amount of damaged cells in the
culture.

11. BASED ON CELLULAR RESPIRATION
• Respiration of the cells measured by oxygen utilization or carbon dioxide
production can be used to assess cell viability. This is usually done by using
Warburg manometer.

12. BASED ON RADIOISOTOPE INCORPORATION
• By using radiolabelled substrates or metabolites, the radiolabel in the products
formed can be detected. This method is particularly useful for the cytotoxicity
assays of drugs.
Labelled nucleotides - Incorporation of (3H) thymidine into DNA and (3H) uridine
into RNA are widely used for the measurement of drug toxicity.
Labelled phosphate- The cells are pre-labelled with 32P. When the damage occurs to
cells they release labelled phosphate which can be measured. The efficacy of drugs
can be evaluated by this approach.

13. BASED ON LUMINESCENCE TEST
• The viability of cells can be measured with good sensitivity by estimating ATP
levels by luminescence based test. The principle is based on the following reaction.

14. TETRAZOLIUM REDUCTION ASSAYS- MTT ASSAY
• The tetrazolium reduction assays measure some aspect of general metabolism
or an enzymatic activity as a 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 viable eukaryotic cells.
Principle: Viable cells with active metabolism convert MTT into a purple
colored formazan product with an absorbance maximum near 570 nm. 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.

15. The MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) tetrazolium
(MTT) substrate is prepared in a physiologically balanced solution.
Added to cells in culture, usually at a final concentration of 0.2 -0.5mg/ml
Incubated for 1 to 4 hours
The quantity of formazan (presumably directly proportional to the number of viable cells) is
measured by absorbance at 570 nm using a plate reading spectrophotometer
Note : The formazan product of the MTT tetrazolium accumulates as an insoluble
precipitate inside cells as well as being deposited near the cell surface and in the culture
medium. The formazan must be solubilized prior to recording absorbance readings.
Various solubilization methods include using: acidified isopropanol, DMSO,
dimethylformamide, SDS.
METHODOLOGY