MC1

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.

This protocol evaluates hepatotoxicity using complementary in vivo mouse APAP acute liver injury and in vitro hepatocyte-based cytotoxicity readouts. In vivo APAP injury is assessed by serum ALT/AST, liver histology, hepatic glutathione, protein adducts, necrosis, inflammation, and regeneration-related endpoints. In vitro hepatotoxicity is assessed by loss of viability, leakage of ALT/AST/LDH, oxidative-stress markers, mitochondrial function, nuclear morphology, intracellular calcium, and high-content imaging endpoints.

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.

Alzheimer’s Disease (AD) is a neurodegenerative disorder characterized by a progressive decline in cognitive functions and loss of specific types of neurons and synapses. Alzheimer's symptoms can be simulated in mice by injecting drugs (such as Aβ) or genetically modified.