LDH Cytotoxicity Assay Kit #37291
Product Information
Protocol
Product Description
The LDH Cytotoxicity Assay Kit can be used for quantitatively measuring cytotoxicity in response to chemical compounds as well as assaying cell-mediated cytotoxicity using a coupled two-step, colorimetric reaction. In the first step, lactate dehydrogenase (LDH) catalyzes the reduction of NAD+ to NADH and H+ by oxidation of lactate to pyruvate. In the second step of the reaction, diaphorase uses the newly-formed NADH and H+ to catalyze the reduction of a tetrazolium salt (INT) to highly-colored formazan, which absorbs strongly at 490-520 nm. The amount of formazan produced is proportional to the amount of LDH released into the culture medium as a result of cytotoxicity.
In a prototypical cytotoxicity assay, target cells are cultured with a cytotoxic chemical agent or a cytotoxic cell (e.g., NK cells) to induce target cell death and LDH release. The LDH-containing supernatants are transferred to wells of a new assay plate and mixed with the LDH Reaction Solution. After an incubation of 30 minutes at room temperature, the absorbance at 490 nm (A490) is read using a plate reader. Cells treated with cytotoxic agents or cytotoxic cells will release an amount of LDH that falls between the maximum release control level and the spontaneous release control level.
In a prototypical cytotoxicity assay, target cells are cultured with a cytotoxic chemical agent or a cytotoxic cell (e.g., NK cells) to induce target cell death and LDH release. The LDH-containing supernatants are transferred to wells of a new assay plate and mixed with the LDH Reaction Solution. After an incubation of 30 minutes at room temperature, the absorbance at 490 nm (A490) is read using a plate reader. Cells treated with cytotoxic agents or cytotoxic cells will release an amount of LDH that falls between the maximum release control level and the spontaneous release control level.
Background
Cell death can occur either by apoptosis, a highly regulated biochemical pathway involving signal transduction cascades, or by necrosis. Necrosis is accompanied by mitochondrial swelling and increased plasma membrane permeability, while apoptosis involves an articulated breakdown of the cell into membrane-bound apoptotic bodies (1). There are a number of assays that are designed to measure cytotoxicity and cell death, independent of mechanism. Most of these assays assess cell viability by measuring plasma membrane permeability (2).
Lactate dehydrogenase (LDH) is a stable, soluble enzyme located in the cytosol of many different cell types. The enzyme is released into the surrounding culture medium upon plasma membrane damage. LDH activity in the culture medium can, therefore, be used as a reliable indicator of cell membrane integrity, and thus a measurement of cytotoxicity.
Lactate dehydrogenase (LDH) is a stable, soluble enzyme located in the cytosol of many different cell types. The enzyme is released into the surrounding culture medium upon plasma membrane damage. LDH activity in the culture medium can, therefore, be used as a reliable indicator of cell membrane integrity, and thus a measurement of cytotoxicity.
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For Research Use Only. Not For Use In Diagnostic Procedures.
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