In vitro cytotoxicity, cell viability, apoptosis

Analysis of cytotoxicity is needed in various biological fields, including toxicology, drug discovery and ecotoxicology where the toxic effects of chemicals, new drugs or environmental samples. The in vitro cell viability (cytotoxicity) assays are used to refrain the unneeded use of animal testing, in vitro assays also being faster and cheaper to conduct, therefore suitable for testing of large number of different compounds and samples.

The mechanisms of cell death include apoptosis and necrosis. The amount of cell death induced by different agents can be measured on various methods.

Respiratory chain activity – MTT assay

MTT assay is a sensitive quantitative colorimetric assay measuring the reduction of yellow tetrazolium salt MTT to dark purple formazan by an enzyme succinate dehydrogenase, mainly in mitochondria. Reduction of MTT to formazan happens only in metabolically active cells and thus can be used to measure the viability of the cells.

Cell membrane integrity – Lactate dehydrogenase assay

Lactate dehydrogenase (LDH) cytotoxicity assay measures the activity of a cytosolic LDH enzyme. The studied substance is applied on the cells and increased LDH activity correlates with increased cell wall damage and LDH leakage to extracellular space, thus acting as an indicator of cell membrane integrity. Like in MTT assay, tetrazolium salt is involved in the colorimetric reaction where LDH activity leads to formation of formazan which is then detected spectrophotometrically.

Lysosomal activity – Neutral read assay

The neutral red (NR) assay is based on the ability of viable cells to incorporate and bind neutral red within lysosomes. Neutral red penetrates cell membrane and accumulates in lysosomal matrix. Changes on the cell surface of the sensitive lysosomal membrane lead to lysosomal fragility and inability to uptake and store neutral red. Neutral red assay is measured quantitatively with a spectrophotometer.

Apoptosis assay

Apoptosis is defined as programmed cell death, characterized by changes including nuclear and cytoplasmic condensation, membrane budding, internucleosomal DNA fragmentation and formation of apoptotic bodies.

Our apoptosis assay is based on assessment of different apoptotic signatures, including caspase activation pathways, DNA fragmentation, LDH release and MTT activity.