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Glycolysis is a major metabolic pathway for producing energy, in which glucose is broken down in the cytosol to produce L-lactate.1) Compared with normal cells, glycolytic activity has been reported to be increased in cancer cells.2) As the main metabolic product of glycolysis, L-lactate is considered an important indicator of the cellular metabolic state. Consequently, quantifying L-lactate concentration is a common method used to evaluate alterations to metabolic pathways in cancer research and related fields.3)
1 enables simple quantification of L-lactate in both cell culture supernatants and intracellular samples. A standard curve is generated using the supplied lactate standard solution, enabling lactate concentrations in samples to be calculated. 1 can measure concentrations from 0.02 to 1 mM; samples with concentrations exceeding 1 mM can be measured after appropriate dilution. This assay quantifies L-lactate concentration based on the absorbance change (at 450 nm) in the WST-8 reduction reaction catalyzed by NADH generated via the L-lactate dehydrogenase reaction (Figure A). Using 1 to evaluate cellular metabolic status, inhibition of glucose uptake by phloretin treatment resulted in a decrease in extracellular L-lactate concentration (Figure B). Conversely, inhibiting mitochondrial electron transport chain complex I with rotenone led to an increase in extracellular L-lactate concentration (Figure B). These results demonstrate that 1 is useful for evaluating changes in L-lactate concentration that reflect alterations in glycolytic activity and L-lactate transport.
Experimental conditions
Cells: HeLa cells (5.0 × 104 cells)
Drug treatment duration: 24 h
Drug concentrations: phloretin, 100 µM; rotenone, 25 µM
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