Lactate assay kit

2 × 10⁴ indicated HEI-OC1 cells per well were seeded into 24-well plates. When cells had reached 60% confluence, the medium was replaced with 1 ml fresh complete medium per well and supernatants were collected after 24 h. The concentration of lactate in supernatant was determined with a lactate assay kit (KeyGEN, China). Briefly, 20 µl supernatant was mixed with the reaction system in 5 ml centrifuge tube. Centrifuge tubes were incubated in a waterbath at 37°C for 10  min, and then 2 ml stop solution was added to reaction system immediately. Finally, the absorbance of the colored substance was measured at 530 nm, and the OD values had a linear relationship with the content of lactate. In particular, OD values of samples between 0.05 and 0.35 are acceptable according to the manufacturer’s protocol of the lactate assay kit. Additionally, protein quantitation was measured by the BCA assays and lactate release counts were normalized to total protein concentrations (mmol/d/g protein). All experiments were performed with five replicates.

Cells were transfected with the indicated shRNA expression vector or overexpression vector and selected using puromycin as previously described. The amount of glucose consumed and the amount of lactate produced in the culture medium were analyzed using the Glucose Colorimetric Assay Kit (BioVision, Milpitas, USA) and the Lactate Assay Kit (KeyGen Biotech, Jiangsu, China), respectively, according to the manufacturer's instructions. The enzymatic activity of G6PD and intracellular NADPH/NADP⁺ levels were detected using the G6PD Assay Kit (Yuanye, Shanghai, China) and AmpliteTM Colorimetric NADPH/NADP⁺ Ratio Assay Kit (Comin Bio, Suzhou, China), respectively, according to the manufacturer's instructions. The total amount of protein determined using the BCA Protein Assay Kit (Beyotime Biotechnology) was used for normalization of the results obtained.

After the transfection of RL95-2 and Ishikawa cells, cells were treated with non-serum medium and starved for 24 h. Then, the cell culture medium was collected to detect the lactate production using a Lactate Assay Kit (KeyGen, Nanjing, China). Lactate production was normalized on the basis of the total protein concentration.