Measurement of cellular 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) reduction activity and lactate dehydrogenase release using MTT

Abstract

3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) reduction assay and lactate dehydrogenase (LDH) release assay have been widely used for evaluating cell viability in culture. MTT reduction assay measures the redox activity of living cells, while LDH assay measures the activity of LDH released into the medium from dead cells. In this paper, we introduce a quick and simple method of measuring cellular MTT reduction and LDH release with the same dye, MTT. The substrate mixture for measuring LDH activity contained lactate, beta-nicotinamide adenine dinucleotide, 1-methoxyphenazine methosulfate, MTT and Triton X-100. When the medium containing LDH was mixed with the substrates, MTT was converted into MTT formazan in proportion to LDH activity. This method was successfully applied for evaluating t-butyl hydroperoxide toxicity in cultured rat cortical astrocytes and glutamate toxicity in cultured rat hippocampal neurons. Our method is economical and convenient especially for measuring cellular MTT reduction and LDH release in the same culture.