LDH Cytotoxicity Assay Kit (BA0001)

LDH Assay Kit - Information

The colorimetric LDH Cytotoxicity Assay Kit is a simple and robust method to assess cytotoxic effects on cells by measuring the activity of LDH in cell culture supernatant. This assay measures cell death in the form of apoptosis and necrosis in response to cytotoxic reagents resulting the release of LDH into the cellular space.

Applications

For rapid quantitative determination of cytotoxicity based on lactate dehydrogenase released into cell culture medium. Evaluation of toxic compounds, toxins, detergents, environmental pollutants and physical treatment on cell lysis.

LDH Assay Kit - Key Features

• Non-radioactive assay (cf. chromium release assay).
• The whole procedure take 20 min.
• Robust and amenable to HTS.
• Single reagent, "mix-incubate-measure" type assay.
• High-throughput assay in 96-well plates allows simultaneous processing tens of thousands of samples per day.

LDH Assay - Data Sheet
Includes	Reagent: 20 mLTriton X-100: 1 mL 20%
Kit Requires	Microplate reader, flat-bottom clear 96-well plate compatible with spectrophotometer, multichannel pipette
Method of Detection	OD500nm
Samples	Cell culture
Species	All
Protocol length	20 min
Size	100 tests
Storage	2-8°C
Shelf Life	12 months after receipt

More Details

Lactate dehydrogenase (LDH) assay can be performed in order to quantitatively determine cell cytotoxicity in the form of tissue or cell damage.LDH is released into the cellular environment when the integrity of the cellular membrane is no longer maintained. Loss cell membrane integrity can occur in the presence of cytotoxic compounds which can induce apoptosis or necrosis. Upon release LDH catalyses the reduction of NAD⁺ to NADH and H⁺ through the oxidation of lactate to pyruvate. Diaphorase a ubiquitous flavin-bound enzyme that catalyzes the reduction of INT which acts as hydrogen acceptors from the reduced form of di- and tri- phosphopyridine nucleotides, i.e., NADH, NADPH. The newly formed NADH and H⁺ catalyse the reduction of tetrazolium salt (INT) to a highly coloured formazan dye with optimum absorbance at 500nm for this LDH assay. The amount of formazan produced is proportional to the amount of LDH released in the media as a result of cytotoxicity.

LDH

Lactate dehydrogenase(LDH) is an oxidoreductase which catalyzes the interconversion of lactate and pyruvate. LDH is a stable cytosolic enzyme that upon membrane damage is released into the cellular environment. Therefore, LDH is often measured to evaluate the presence of tissue or cell damage. The assay is based on the reduction of a tetrazolium salt to a formazan dye.

Tetrazolium Salts and Formazan Dyes

Tetrazolium salts are widely used in cell biology and clinical biochemistry for measuring the metabolic activity of mammalian and microbial cells. Major tetrazolium salts include MTT, INT, TTC, MTS, XTT and NBT. These dyes are mainly heat and light unstable and can be reduced dehydrogenases, reductases and other reducing agents into Formazan dyes. INT is mainly used for LDH assays.Formazan dyes display a broad spectrum of colors from dark blue, deep red, to orange, depending on the original tetrazolium salt used for reaction. Second generation tetrazolium dyes such as XTT and MTS form water-soluble formazans and require an intermediate electron acceptor for reduction.