Enzychrom glycolysis assay kit

Cell culture medium RPMI1640, fetal calf serum (FCS), antibiotics, and trypsin were from Invitrogen (Waltham, MA, USA). LPA (1-oleoyl-2-hydroxy-sn-glycero-3-phosphate; LPA18:1) was from Sigma-Aldrich (St. Louis, MO, USA). Antibodies were from Cell Signaling Technology (Danvers, MA, USA), Abcam (Cambridge, UK) and Santa Cruz Biotechnology Inc. (Dallas, TX, USA) as listed in Table 1. ROS assay kit was ROS-ID^® Total ROS Detection Kit (ENZO Life Sciences, Farmingdale, NY, USA). Glutathione assay kit was GSH-Glo™ Glutathione Assay Kit (Promega Corporation, Madison, WI, USA). Lactate assay kit was EnzyChrom™ Glycolysis Assay Kit (Bioassay Systems, Hayward, CA, USA). Primers were from Qiagen (Hilden, Germany) and Invitrogen (Waltham, MA, USA). Protein estimation kit was Pierce™ BCA Protein Assay Kit (Thermo Fisher Scientific, Waltham, MA, USA).

L-lactate levels in the brain and muscle were measured in the indicated genotypes. L3 larval brains and muscle carcasses were dissected and placed in ice-cold 1X PBS. Brains (n = 20 each, total 60 brains per genotype) or muscle tissue (N = 8, total 24 carcasses per genotype) were pooled together and homogenized in 50 μL 1X PBS. Bradford Assay was used to quantitate protein concentrations. Each lysate was transferred to a 96-well plate and incubated with lactate dehydrogenase and NAD/MTT for 2 hr at room temperature (EnzyChrom Glycolysis Assay Kit, BioAssay Systems). The intensity of the reduced dye was measured at 565 nm, which is directly proportional to the concentration of the L-Lactate in the sample.