L lactate assay kit ab65330

Fetal bovine serum (FBS) was obtained from Life Technologies (Waltham, MA, USA). RPMI and MEM medium were purchased from Welgene Inc. (Gyeongsan, Korea). The antibodies against PKM1 (21577) and PKM2 (21578) were obtained from Signalway (Greenbelt, MD, USA). Lamin B1 antibody (12586), horseradish peroxidase (HRP)-conjugated goat anti-mouse (7076) and anti-rabbit secondary antibodies (7074) were obtained from Cell Signaling Technology (Danvers, MA, USA). β-actin antibody (A2228), vinculin antibody (V9131), and all the chemicals, including DMSO (D8418), gefitinib (GE, SML1657), dextran sulfate (D8906), vanadyl-ribonucleoside complex (94742), E. coli RNAse-free tRNA (10109541001), and formamide (221198), were purchased from Sigma-Aldrich (St. Louis, MO, USA). The L-Lactate assay kit (ab65330) and the extracellular oxygen consumption assay (ab197243) were purchased from Abcam (Cambridge, UK). 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) was purchased from Invitrogen (Waltham, MA, USA).

The lactate levels in the cell supernatants were measured after 24 hours of culturing under the conditions described above using an L-lactate assay kit (ab65330) (Abcam Cambridge, United Kingdom) following the manufacturer’s instructions. Briefly, after the samples were deproteinized, each sample was incubated with lactate assay buffer, lactate substrate mix, and lactate enzyme mix in a 96-well plate. After incubating for 30 minutes at room temperature, the optical density was measured at 450 nm with a microplate reader.