Basic green 4

A carcinogenicity bioassay detects whether long-term exposure to a test substance increases benign or malignant tumor incidence, changes tumor spectrum, or shortens tumor latency in experimental animals; the classical rodent design exposes rats and/or mice to multiple dose levels for most of their lifespan, followed by complete necropsy and histopathologic diagnosis of neoplastic and non-neoplastic lesions. The readout is tumor incidence by organ, sex, species, dose group, and survival status; interpretation requires concurrent controls, dose-response assessment, survival-adjusted tumor statistics, and pathology review because mortality, spontaneous tumor background, and body-weight effects can influence apparent tumor rates.

Cytotoxicity assays are usually based on the assessment of cell membrane damage, which can also be indirectly detected by measuring cell viability. Detection methods include MTT assay, CKK-8 assay, LDH assay and ATP assay, etc.

Live/dead viability and cytotoxicity staining assays are based on the simultaneous detection of intracellular esterase activity in metabolically active (viable) cells and membrane integrity loss in non-viable cells. In commonly used dual-staining approaches, membrane-permeant fluorogenic substrates are converted by intracellular esterases into fluorescent products in live cells, while impermeant DNA-binding dyes selectively enter cells with compromised plasma membranes and label nucleic acids in dead or dying cells, enabling discrimination between viable and non-viable populations by fluorescence microscopy or flow cytometry.

The following protocol uses the MTT colorimetric assay as a classic literature-established method for assessing cell viability/metabolic activity in cultured mammalian cells. MTT[3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] is reduced by metabolically active cells to a colored formazan product; the amount of formazan is quantified spectrophotometrically and provides an indirect measure of metabolically active viable cells. Importantly, MTT reduction reflects cellular oxidoreductase/metabolic activity rather than an absolute direct count of living cells, so changes in cellular metabolism can alter the signal independently of cell number.