ASSIGNMENT-1 ADIL ARSHAD

BMI-19001

1. Explain Viability assay, Survival assay, Microtitration assay,

Transformation assay with an example.

ANS- 1. Cytotoxicity and Viability Assays:

A majority of the cytotoxicity and viability assays are based on the measurement of
membrane integrity, cellular respiration, radioisotope incorporation, colorimetric
assays and luminescence- based tests.

Based on membrane integrity:

The most common measurements of cell viability are 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) or release of dyes normally taken up and retained by viable
cells (e.g. neutral red, diacetyl fluorescein).

The other assays for membrane integrity are release of labeled chromium (51Cr),
enzymes and use of fluorescent probes. Cell viability measurements, based on
membrane integrity are immediate that can be detected within a few hours.
However, these measurements cannot predict the ultimate survival of cells.

2. Survival Assays:
The tests described above for measurement of cell viability and cytotoxicity are
short-term, and they identify the dead/live cells at the time of assay. Many times,
when the cells are subjected to toxicity (i.e. exposed to drugs, irradiated), the
effects are not immediate, but may be observed after several hours or sometimes
even days. The assays based on the survival of cells (i.e. retention of regenerative
capacity or reproductive integrity) are preferred.
 3. MTT-based cytotoxicity assay is carried out in the following stages:

1. Incubation of monolayer cultures with varying drug concentrations in micro-

titration plates.

2. Removal of drug and feeding of plates to achieve 2-3 PDTs.

3. Treatment of plates with MTT, and removal of medium and MTT.

4. Measurement of MTT-formazan in an ELISA plate reader.

When the absorbance of test wells/control wells of the micro-plate is plotted

against the concentration of the cytotoxic drug, a sigmoid curve is obtained.

iv. Transformation Assays:

The following are the commonly used assays for measurement of in vitro
transformation:
i. Evidence of mutagenesis.
ii. Anchorage independence.
iii. Reduced density limitation of cell proliferation.
Mutagenesis:
Mutagenesis can be assayed by sister chromatid exchange (SCE). SCE basically
involves the reciprocal exchange of DNA segments between sister chromatids at
identical loci in the S-phase of cell cycle. Sister chromatid exchanges are more
sensitive to mutagenesis than chromosomal breaks. For this reason, SCEs are
preferred in mutagenesis research and transformation assay. The SCE technique
basically involves the incorporation of radioactive nucleotides into replicating
DNA and detection of SCEs by fluorescence plus Giemsa (FPG) technique.