ShK toxin

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.

Primary dorsal root ganglion sensory neuron culture isolates DRG neuronal somata from rodent or human ganglia, dissociates tissue enzymatically and mechanically, and maintains post-mitotic sensory neurons in vitro for readouts such as neurite outgrowth, immunocytochemical marker expression, calcium imaging, electrophysiology, RNA/protein analysis, or neuropeptide release assays. The method reflects peripheral sensory neuron biology because DRG neurons are primary sensory neurons whose cell bodies reside in dorsal root ganglia and whose cultured dissociated cells can retain neuronal morphology, sensory-neuron marker expression, and stimulus-responsive properties depending on the downstream assay.

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.