Invitrogen™ CyQUANT™ LDH Cytotoxicity Assay, fluorescence

Description

• 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.