Four 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 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.

Glucose test principle

Many cultured cells continually consume glucose from their culture media for the generation of biosynthetic intermediates. By knowing the initial glucose concentration in the media, its consumption rate may be easily assessed by monitoring glucose levels in the culture medium after incubation in the absence and presence of a test compound. Differences of glucose consumption in treated and untreated cells reflect changes in their metabolic state after drug exposure.

The assay allows to determine the physiological state of cultured cells by measuring glucose consumption. This procedure utilizes the coupled activities of glucose oxidase (GOD) and peroxidase (POD):



The quantity of oxidized dye in the supernatant is measured spectrophotometrically at 540 nm. This method is suitable for end point and kinetic techniques.

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 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 soluble tetrazolium salt into a formazan dye 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. An increase in the number of living cells results in an increase of total metabolic activity which leads to a stronger color formation.

SRB test principle

Cell proliferation, measured as total protein synthesis, is a very sensitive toxicology marker. Sulforhodamine B (SRB, Acid Red 52) is an anionic dye that binds electrostatically to cellular proteins. The fixed dye is solubilized and is measured photometrically at OD 540 nm with a reference filter of 690 nm. The OD values correlate up to 1.8 OD units with total protein synthesis rate and therefore with cell proliferation.

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