Principle of Assay
Lactate dehydrogenase (LDH) is an oxidoreductase which catalyzes the interconversion of pyruvate and lactate with concomitant interconversion of NADH and NAD+. LDH is a stable enzyme, present in all cell types, and rapidly released into the cell culture medium upon damage of the plasma membrane. LDH, therefore, is the most widely used marker in cytotoxicity studies. This kit provides a simple and easy colorimetric assay for the study of cytotoxicity. This assay is based on the reduction of the tetrazolium salt MTT in a NADH-coupled enzymatic reaction to a reduced form of MTT which exhibits an absorption maximum at 565 nm. The intensity of the generated color correlates directly with the number of cells lysed. In a typical cytotoxicity assay, target cells are cultured with a cytotoxic chemical agent or a cytotoxic cell (NK cell, cytotoxic T cells) to induce target cell death and LDH release. The LDH-containing supernatants are transferred to wells of a new 96-well assay plate and mixed with the LDH Reaction solution. After an incubation of 30 minutes at room temperature, the absorbance at 565 nm is read using a plate reader.
