Glutamate pyruvate transaminase

The amount of cellular lactate release was measured as described previously^{30 (link)}. Briefly, the supernatant from cultured cells was de-proteinized with perchloric acid and neutralized with potassium hydroxide. The supernatant was mixed with nicotinamide adenine dinucleotide and glutamate pyruvate transaminase (Roche, Mannheim, Germany). The enzymatic reaction was started by adding lactate dehydrogenase (Wako) to each sample and incubating at 37 °C for 30 min. Absorbance was measured at a wavelength of 340 nm.

Release of cellular lactate was measured as described previously [21 (link)]. Briefly, the supernatant from cultured cells was de-proteinized with perchloric acid and neutralized with potassium hydroxide. The supernatant was then mixed with nicotinamide adenine dinucleotide and glutamate pyruvate transaminase (Roche, Mannheim, Germany). The enzymatic reaction was started by adding lactate dehydrogenase (Wako) to each sample at 37°C for 30 min. Absorbance was the measured at a wavelength of 340 nm.

Cell Supernatants were collected and directly frozen after culture experiments. L-lactate levels were measured using two enzymatic reactions. Lactate dehydrogenase (LDH; Roche, Meylan, France) catalyzed the NAD⁺ -mediated oxidation of lactate into pyruvate. Then glutamate-pyruvate transaminase (GPT; Roche, Meylan, France) was used to shift first reaction equilibrium by transforming all the pyruvate into alanine and α-ketoglutarate. The amount of NADH formed was related to the amount of lactate processed by these two coupled reactions. Briefly, 20 μL of each sample were added to 200 μL of reaction buffer (620 mM sodium carbonate, 78.7 mM L-glutamate, 0.92 mM NAD, 2 μg GPT and 2 μg LDH). A standard curve was obtained with lithium lactate (Sigma Aldrich). 96 multiwell plates were incubated at 37 °C for 30 minutes before quantifying extracellular lactate production by monitoring the increase in absorbance of NADH at 355 nm on a spectrophotometer (SpectraMax Gemini; Molecular Devices, France). At least three independent experiments, performed in triplicate and normalized to the protein content, were carried out for each experimental condition.