6.2.4 Lactate dehydrogenase (LDH) assay
A colorimetric method for assessing cellular cytotoxicity, the LDH
cytotoxicity assay can be used with a variety of cell types to examine
cytotoxicity brought on by hazardous chemicals and other test substances
as well as cell-mediated cytotoxicity. The assay detects the stable,
cytosolic LDH enzyme, which is released from damaged cells and
quantified using a linked enzymatic reaction that turns
iodonitrotetrazolium (INT) into a red-colored formazan through the
action of the diaphorase. During the initial stage of the procedure, by
reducing NAD to NADH/H+, LDH catalyses the conversion of lactate to
pyruvate. The catalyst (diaphorase) moves H/H+ from NADH/H+ to the
tetrazolium salt w2-(4-iodophenyl)-3-(4-nitrophenyl)-5-phenyltetrazolium
chloride (INT) in the second step, where it is reduced to red formazan.
The rate of INT reduction or NADH oxidation is used to measure LDH
activity over time. This causes a red fluorescence that can be measured
at 490 nm. In the LDH experiment, the detergent Triton X-100 is
frequently used as a positive control to assess the maximum LDH release
from the cells. Positive controls, such as crystalline silica, a
well-known membranolytic particle, can also be used.(Schins et al.,
2002). This test has several advantages, including reliability,
rapidity, and ease of evaluation.
The inherent LDH activity of blood and several other medium components
is a significant flaw in this test. For instance, the background
concentrations in foetal calf serum are extremely high. This assay is
restricted to serum-free or low-serum conditions, limiting the assay’s
culture length and breadth. This is due to the assay’s inability to
detect cell death produced under standard growth conditions with 10%
foetal calf serum in the media. Always compare readings from unused
study medium portions to readings from additive-free media, like DMEM
(Fotakis and Timbrell, 2006).