The Thermo Scientific Pierce LDH Cytotoxicity Assay Kit is a reliable colorimetric assay to quantitatively measure lactose dehydrogenase (LDH) released into the media from damaged cells as a biomarker for cellular cytotoxicity and cytolysis.
The Pierce LDH Cytotoxicity Assay Kit measures extracellular LDH in culture media using an enzymatic reaction that results in a red formazan product which can be measured spectrophotometrically. Lactate dehydrogenase (LDH) is a cytosolic enzyme that is is an indicator of cellular toxicity. The assay is ideal for high-throughput screening and provides a safe alternative to radioactive cytotoxicity assays.
- Convenient – add-mix-read assay format for adherent and suspension cells
- Colorimetric – quantitatively measures LDH release by formation of colored product
- Robust – uses stable LDH enzyme activity as a cytotoxic marker
- Flexible – ideal for high-throughput screening
- Non-radioactive – safe alternative to 51Cr-release cytotoxicity assays
Kit contains substrate mix, assay buffer, 10X lysis buffer, stop solution, and LDH positive control
Microplate reader capable of reading absorbance at 490nm and 680nm, flat-bottom clear 96-well plate compatible with spectrophotometer, multichannel pipette
- Measure in vitro cytotoxicity mediated by chemicals, immune cells, or siRNA or microRNA
- Detect cytolysis in bioreactor or 3D tissue engineering applications
Lactate dehydrogenase (LDH) is a cytosolic enzyme present in many different types of cells. When the plasma membrane is damaged, LDH is released into cell culture media. The released LDH can be quantified by a coupled enzymatic reaction. First, LDH catalyzes the conversion of lactate to pyruvate via reduction of NAD+ to NADH. Second, diaphorase uses NADH to reduce a tetrazolium salt (INT) to a red formazan product. Therefore, the level of formazan formation is directly proportional to the amount of released LDH in the medium.
|Metabolic and chemical basis of the LDH cytotoxicity assay.
The assay is performed by transferring cell culture media from treated cells into a new microplate and adding the kit reagents. After incubation at room temperature for 30 minutes, reactions are stopped and LDH activity is determined by spectrophotometric absorbance at 490nm.
|Protocol summary for Thermo Scientific Pierce LDH Cytotoxicity Assay Kit.
|Determination of Actinomycin D-induced cytotoxicity in CHO-K1 cells using the Thermo Scientific Pierce LDH Cytotoxicity Assay. CHO-K1 cells were plated in a 96-well plate (10,000 cells per well) in RPMI1640 medium supplemented with 10% fetal bovine serum and incubated overnight at 37°C in 5% CO2. Cells were then treated with different concentrations of the cytotoxic agent, Actinomycin D, for 18 hours. Actinomycin-induced cytotoxicity was measured using the Pierce LDH Cytotoxicity Assay Kit (red; left y-axis). Cell viability was measured in replicate wells using the alamarBlue Cell Viability Reagent (Part No. 88951, 88952)(blue; right y-axis).
- Decker, T. and Lohmann-Matthes, M.L. (1988). A quick and simple method for the quantitation of lactate dehydrogenase release in measurements of cellular cytotoxicity and tumor necrosis factor (TNF) activity. J. Immunol. Meth. 115:61-9.
- Korzeniewski, C. and Callewaert, D.M. (1983). An enzyme-release assay for natural cytotoxicity. J. Immunol. Meth. 64:313-20.
alamarBlue Cell Viability Assay Reagent
Cellular Imaging and Analysis Reagents (formerly Cellomics Products)