DG013A

By lysing cells, releasing RNA, and removing impurities such as proteins and DNA, high-purity RNA products are finally obtained. The commonly used traditional method is the guanidine isothiocyanate/phenol/chloroform method (Trizol), which is suitable for a variety of animal materials including animal tissues, microorganisms, cultured cells, etc., and most plant materials.

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.

Serum-free B27/Neurobasal culture is a defined neuronal maintenance method designed to support dissociated primary neurons while limiting serum-driven glial expansion; the readout is sustained neuronal survival, neurite extension, neuronal marker expression, synapse formation, and, when measured, electrophysiological or calcium activity. B27/Neurobasal was optimized in embryonic rat hippocampal neurons, where B27 supported >60% survival after 4 days above 160 plated cells/mm² and Neurobasal reduced glial growth to <0. 5% by immunocytochemistry; later studies extended the approach to cortex, striatum, substantia nigra, septum, cerebellum, and dentate gyrus neurons.

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.