Cellular models of Parkinson’s Disease are useful for testing the efficacy of intrabodies (intracellular antibodies) against the pathological effects of alpha–synuclein (protein molecule implicated in the disease) and its mutants, when transiently transfected into the cells. A marker indicative of improved cell viability is cell toxicity rescue.
A widespread technique for measuring cell death is to quantify the release of lactate dehydrogenase (LDH), a stable enzyme found in the cytoplasm, into the surrounding media upon rupture of the plasma membrane. Most LDH cytotoxicity assays are colorimetrically based, and typically measure the absorbance of a product, the result of a reaction between a supplied reagent and the LDH in media, at a particular wavelength. The absorbance correlates positively with the number of compromised cell membranes.
This BioVision version of this assay uses the advanced tetrazolium WST reagent to react with NADH produced by lactate from LDH, to give an intense yellow color directly correlating with the amount of LDH in the media. The kit also comes with a vial of LDH for positive controls, LDH assay buffer, cell lysis solution, and stop solution. Typically adherent cells are grown in a 96-well plate, under user-specified conditions with additional high toxicity and low toxicity controls. After a user-defined period of incubation to allow for accurate determination of toxicity properties, 10 µls of lysis solution are added to the high controls for 30 min. Aliquots are then removed from each well for LDH analysis with WST reagent (which has been previously reconstituted and diluted in assay buffer). Subsequently, the plate is incubated at room temperature in the dark for 30 min., and the absorbance measured at 450 nm with reference wavelength 650 nm. Typically, OD450 of high control reads about 2.0, and those of low control should read < 0.8. The reaction can be stopped by adding 10 µl of stop solution, mixing, and reading within 48 hrs.
In most assays, the signal obtained is compromised by high background noise from the serum in the media, thus requiring the use of serum-free culture. Lack of serum in media results in compromised cell growth unless a growth factor or supplement is added, and may result in non-comparable results. The advantage of the WST used in the BioVision assay is that it generates intense signals which essentially reduce the background obtained from serum. This allows the use of 10% serum-medium in the assay protocol, and gives consistent results in my experience, when 1e-4 cells/well or fewer are used. At higher cell densities (ST14A rat cells), I have found readings to be unreliable. The compound has been reported to be stable, and the reaction can be stopped and read up to 48 hours later. The assay is purported to take only one hour; however, if the high controls are performed, completion can take up to an extra 30 minutes. Other than this, my experience with this kit has been satisf actory and reports reproducible results in the presence of media with 10% fetal bovine serum.
Postdoctoral Research Affiliate
Division of Genetic Disorders
Health Research Incorporated