L lactate assay kit

Left ventricular tissues were collected by snap-freezing and homogenized in PBS at 4°C. The homogenized lysates were centrifuged at 12,000g at 4°C to collect supernatant. The primary cardiomyocytes were cultured in M199 with indicated treatments, and culture medium was collected. Lactate concentration was determined using an L-Lactate Assay Kit (Trinity Biotech, 736-10). Intracellular NAD(H) level and glutathione were determined by an EnzyChrom NAD⁺/NADH Assay Kit (Bioassay Systems, E2ND-100) and a GSH/GSSG Ratio Detection Assay Kit (Abcam, ab239709), respectively, according to the manufacturers’ protocols. To assay glycogen level, hearts were harvested 7 days after MI or sham surgery. Mice were fasted for 8 hours before tissue harvesting. Ventricular tissues were quickly excised and briefly washed with cold 0.9% NaCl before snap-freezing. Glycogen was separated from exogenous glucose in 10 mg cardiac tissue by an alkaline extraction procedure and measured using a commercially available kit (BioVision, catalog k960-400).

1.5 × 10⁵ hTERT-BJ1 cells per well were seeded in a 12 well plate. When cells were attached, drug treatments were added in triplicate for 48 or 72 h. Media was then collected. L-lactate was measured using the L-Lactate Assay Kit (735-10, Trinity Biotech) and β-hydroxybutyrate (β-HB) was determined with the β-HB Assay Kit (K632, Biovision), according to the manufacturer. L-lactate and β-HB production were calculated by subtracting the levels of L-lactate or β-HB in complete media from those in each sample. Glucose concentration was quantified using a FreeStyle Optium Glucose Meter (Abbott). Glucose consumption was calculated by subtracting the levels of glucose in each sample from those in complete media. For all assays, viable cells in each well for each time point were counted using Trypan Blue (T8154, Sigma) and used to normalize all results.