The tests to determine the cell viability and cell cytotoxicity have a short term result as they identify the dead or live cells at the time of the assays. Many times when the cells are subjected to toxicity such as exposure to drugs, irradiation etc the effects are not immediate. The effects are observed after several hours or sometime after some days. The assays based on the survival of cells like retention of regenerative capacity or reproductive capacity is preferred.
In clonogenic assay the survival of the cells is measured by the plating efficiency which is the percentage of cells seeded at subculture that give rise to colony. The plating efficiency measures the proliferative capacity for several cell generations. Clonogenic assay broadly consists of the following stages.
1. Treatment of the cells with varying concentration of experimental agents for around 24
2. Trypsinization followed by seeding of cells at low density.
3. Incubation of the cells for one to three weeks.
4. Staining and counting of the clones.
A survival curve based on semi log plot is drawn. It represents the survival fraction of the cells against drug concentration. The inhibitory concentration refers to the drug concentration required to inhibit the minimum viability of cells.
The clonogenic assay is influenced by several factors. The important ones are listed below.
A. Concentration of toxic agents
B. Duration of exposure
C. Cell density during exposure
D. Cell density during cloning
E. Size of colony
MTT Based Cytotoxicity Assay
The tetrazolium salt or 3, (4, 5-dimethyl thiozol-2-yl)-2, 5-diphenyl tetrazolium bromide is commonly known as MTT. It is a dye and is widely used in cytotoxicity assays.
The growing cells in the log phase are exposed to cytotoxic drug. The drug is then removed and the cells are allowed to proliferate for 2 to 3 population doubling times or PDTs. The number of surviving cells can be detected by MTT dye reduction. The concentration of MTT formazan formed can be determined spectrophotometrically in the region of 500 to 600 nm. Formazan is an artificial chromogenic products formed due to reduction of MTT by dehydrogenase and reductase.
MTT based cytotoxicity assay is carried out in the following steps.
1. Incubation of monolayer cultures with varying drug concentrations in micro
2. Removal of drugs 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 the test wells or control wells of the micro concentration plate is plotted against the concentration of the cytotoxicity drug a sigmoid curve is obtained.
The metabolic assays are based on the measurements of metabolic responses of the cells. These tests are carried out after exposure of the cells to cytotoxic drugs or either immediately or after 2-3 population doublings.
The most commonly used metabolic measurements are DNA, RNA or protein synthesis by estimating their concentrations, beside the assay of certain dehydrogenase enzymes.
There are some limitations of the metabolic assays. The estimation of the total content of DNA or protein may or may not be indicative of increase in cell number. This is because these assays cannot discriminate between the proliferative and metabolic activity of cells.
In this type of assays the in vitro transformation is used for measuring the cell viability. The following are the commonly used assays for measurement of in vitro transformation.
• Evidence of mutagenesis
• Anchorage independence
• Reduced density limitation of cell proliferation
Mutagenesis can be assayed by sister chromatid exchange or 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 Giesma technique.
Inflammation assays are required for testing the various forms of allergy induced by cosmetics, pharmaceuticals and other xenobiotics. These assays are at the early stages of development in the culture cells.
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