CyQUANT™ LDH Cytotoxicity Assay, fluorescence

The CyQUANT LDH Cytotoxicity Assay, fluorescence, is a fluorescent assay that provides a simple and reliable method for determining cellular cytotoxicity. Lactate dehydrogenase (LDH) is a cytosolic enzyme present in many different cell types that is released into the cell culture medium upon damage to the plasma membrane. The CyQUANT LDH Cytotoxicity Assay, fluorescence, provides the reagents to accurately and quantitatively measure this extracellular LDH.

The CyQUANT LDH Cytotoxicity Assay, fluorescence, features include:
• Convenient—add-mix-read assay protocol for adherent and suspension cells, including 3D cell models
• Accurate—provides a quantitative measurement of LDH release
• Flexible—ideal for high-throughput screening, monitor cytotoxicity from the same sample over time
• Robust—formulation with highly purified resazurin results in a large assay dynamic range

LDH is a cytosolic enzyme present in many different cell types and is a well-established and reliable indicator of cellular toxicity. Damage of the plasma membrane results in a release of LDH into the surrounding cell culture medium. This extracellular LDH can be quantified by a coupled enzymatic reaction in which LDH catalyzes the conversion of lactate to pyruvate via NAD+ reduction to NADH. Diaphorase then uses NADH to reduce resazurin to resorufin that can be detected using an excitation of 560 nm and emission of 590 nm. The level of resorufin formation is directly proportional to the amount of LDH released into the medium.

As a consequence of the synthesis and manufacturing processes, all resazurin-based reagents contain a detectable amount of resorufin contamination. The amount of contamination can vary greatly between sources of the material and manufacturing conditions, contributing to differences in detectable background fluorescence. More importantly, the contaminating resorufin reduces the signal to background ratio and dynamic range of the assay. An innovative process was developed that removes the contaminating resorufin, resulting in the highly pure resazurin used in the CyQUANT™ LDH Cytotoxicity Assay, fluorescence.

The CyQUANT LDH Cytotoxicity Assay, fluorescence, provides the reagents needed for the simple, reliable fluorescence-based quantification of cellular cytotoxicity. The kit can be used with different cell types, including 3D cell models, to measure cytotoxicity mediated by chemical compounds as well as cell-mediated cytotoxicity. Since LDH in the medium is the indicator of cellular cytotoxicity, the assay can be used to monitor cytotoxicity from the same sample over time. To perform the assay, an aliquot of the cell culture medium is transferred to a new plate and the reaction mixture is added. After a 10-minute incubation, the reaction is stopped by adding Stop Solution and fluorescence is measured using a microplate reader.