Cytotoxicity assays are widely used in the domain of pharmaceutical research. Applications include screening for cytostatic and cytotoxic anti-cancer drugs, measurement of cell proliferation (IC50 values) in response to different drugs, extracts, formulations, growth factors, cytokines, mitogens and nutrients, and assessment of physiological mediators that inhibit cell growth.
Determining the half-maximal inhibitory concentration (IC50) is a common objective of these assays. IC50 is a measure of the effectiveness of a substance in inhibiting a specific biological or biochemical function.
The assays can be conducted on different levels:
- Quick screening of cytotoxic activity: checking viability % of 2 concentrations of the test compound.
- Routine determination of IC50 (using a logarithmic serial dilution): testing 5 concentrations of the test compound.
- Thorough determination of IC50 (using logarithmic & half-log serial dilution): testing 10 concentrations of the test compound.
Available testing protocols
A. Sulforhodamine B (SRB) cytotoxicity assay
Sulforhodamine B (SRB) cell cytotoxicity assay is one of the most widely used methods to detect cell viability or drug cytotoxicity. It is simple, accurate, reproducible and sensitive. It is the method of choice for high cost-effective screenings. This method relies on the property of SRB (a bright-pink aminoxanthene dye), which binds stoichiometrically to proteins under slightly acidic conditions and then can be extracted using basic conditions; thus, the amount of bound dye can be used as a proxy for cell mass, which can then be extrapolated to measure cell proliferation. It can be measured at 565 nm.
This assay is independent of cell metabolic activity and therefore showing less interference by the testing compounds and is one of the most reproducible cytotoxicity assays ever.
B. WST-1 cell proliferation assay
WST-1 assay is an upgrade for MTT and MTS assays, which are colorimetric assays for measuring the activity of enzymes that reduce MTT or structurally related dyes (XTT, MTS, and WSTs) to formazan dyes. The assay is highly convenient as it requires neither washing nor harvesting or solubilization of cells. WST -1 yields water-soluble cleavage products. The generation of the dark yellow colored formazan is measured at 420-480nm (optimal at 440nm) and is directly correlated to cell number.
WST-1 is more stable, has a wider liner range and shows accelerated color development compared to other structures related dyes.