Three Parameter Cytotoxicity Test Kits

IN CYTOTOX products are available worldwide

Extracellular LDH test principle

Cell death or cytotoxicity is classically evaluated by the quantification of plasma membrane damage. Lactate dehydrogenase (LDH) is a stable cytoplasmic enzyme present in all cells, and is rapidly released into the cell culture supernatant upon membrane damage or cell lysis. This assay is a fast and simple method to determine changes in the plasma membrane upon incubation with a test compound

LDH activity reduces pyruvate to lactate by oxidizing NADH to NAD+:



NADH degradation is measured spectrophotometrically by a decrease in the OD at 340 nm.

XTT test principle

Viable cells depend on an intact mitochondrial respiratory chain and an intact mitochondrial membrane. Toxic agents can be identified using mitochondrial dehydrogenases from viable cells.

XTT (2,3-bis[2-methoxy-4-nitro-5-sulfopheny]-2H-tetrazolium-5-carboxyanilide inner salt) is a tetrazolium salt that is cleaved to formazan by the succinate dehydrogenase system which belongs to the mitochondrial respiratory chain, and is only active in viable cells. The mitochondrial succinate dehydrogenase reduces the yellow tetrazolium salt into soluble orange formazan in the presence of an electron coupling reagent.

In contrast to the insoluble formazan salt crystals of MTT, XTT is converted to a water-soluble formazan product without the need for a solubilization step prior spectrophotometric quantification. The enzyme activity is measured at 480 nm (optimum) or at 450 nm.

NR test principle

The neutral red (NR) assay procedure is a cell survival/viability assay, based on the ability of viable cells to incorporate and bind neutral red within lysosomes. It is generally performed on adherent cells. NR is a weak cationic dye that readily penetrates the cell membrane and accumulates intracellularly in lysosomes (lysosomal pH < cytoplasmic pH), where it binds with anionic sites to the lysosomal matrix (G. GRIFFON, et al. 1995). Changes of the cell surface or the sensitive lysosomal membrane lead to lysosomal fragility and other changes that gradually become irreversible. Such alterations brought about by the action of xenobiotics result in a decreased uptake and binding of NR. It is thus possible to distinguish between viable, damaged, or dead cells, which is the basis of the assay.

The quantity of dye incorporated into cells is measured by spectrometry at 540 nm, and is directly proportional to the number of cells with an intact membrane.

The assay can be used to evaluate cytotoxicity by determination of the IC50 (50% inhibiting concentration).

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