Iturin A-2

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.

The CFSE (carboxyfluorescein diacetate succinimidyl ester) dye dilution proliferation assay is based on the covalent labeling of intracellular proteins by a cell-permeant fluorescent dye that becomes fluorescent upon intracellular ester cleavage and then is stably retained within cells. As labeled cells divide, the dye is partitioned equally between daughter cells, resulting in a stepwise halving of fluorescence intensity that can be quantified by flow cytometry to determine the number of cell divisions undergone by each cell population. This fluorescence dilution approach enables quantitative tracking of lymphocyte proliferation at the single-cell level over multiple rounds of division. CFSE-based proliferation analysis has been widely applied to measure antigen-driven lymphocyte expansion in vitro, where discrete fluorescence peaks correspond to successive cell divisions and allow reconstruction of proliferative history within heterogeneous populations.

Dye-dilution cell tracking assays quantify cell proliferation by covalently labeling intracellular proteins with a stable fluorescent dye that is equally partitioned between daughter cells during mitosis, resulting in stepwise halving of fluorescence intensity with each cell division as measured by flow cytometry histograms. Carboxyfluorescein diacetate succinimidyl ester (CFSE) is a prototypical dye that diffuses into cells, is enzymatically converted into a fluorescent compound, and then covalently binds intracellular amine groups, producing long-lived fluorescence suitable for tracking multiple rounds of division in vitro and in vivo. Successive generations of dividing cells form discrete peaks of decreasing fluorescence intensity, enabling estimation of proliferation history, precursor frequency, and division index within heterogeneous populations. Alternative dyes such as CellTrace Violet (CTV) and far-red membrane dyes (e. g. , PKH26) follow the same dilution principle but differ

Breast cancer is a heterogeneous cancer, and it has been distinguished into four subtypes: luminal A, luminal B, HER2-positive and basal-like. Molecular mutations, epigenetic alterations, hormone exposure and immune microenvironment are related to the progression of breast cancer.

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.