Lactate assay kit 2

Hypoxia-treated A549 and H1975 cells were starved in 0.1% glucose-free RPMI containing 0.1% FBS for 16 h. Then, to these cells was added 2-(N-[7-nitrobenz-2-oxa-1,3-diazol-4-yl] amino)-2-deoxy-d-glucose (5 μM) for 30 min, and the fluorescence intensity was read using a microplate reader (Clario Star; BMG Labtech, Ortenberg, Germany). The concentration of lactate was measured using lactate assay kit II (Invitrogen) before collection of the cell extract of hypoxic A549 and H1975 cells.

For the glucose uptake assay, the Glucose Uptake Colorimetric Assay kit was employed (Invitrogen, USA). A total of 2000 cells were seeded in a 96-well plate and treated with Krebs-Ringer-Phosphate-HEPES buffer containing 2% bovine serum albumin (Servicebio, Wuhan, China) for 40 min. Subsequently, 10 mL of 2-deoxyglucose (10 mM) was added to each well. After culture for 20 min, the rate of glucose uptake was measured. For the measurement of lactate production and ATP synthesis, the Lactate Assay Kit II (Invitrogen) and ATP Assay kit (Invitrogen) were used, respectively. A total of 2×10⁶ cells were lysed in 100 μl of the corresponding assay buffer and homogenized. Following centrifugation, the soluble fraction was assayed.

To detect glucose uptake in each group, the Glucose Uptake Colorimetric assay kit (Invitrogen; Thermo Fisher Scientific, Inc.) was used according to the manufacturer's instruction. A total of 2,000 MG63 and Saos-2 cells were seeded in a 96-well plate and treated with the Krebs-Ringer-Phosphate-HEPES buffer containing 2% BSA (Wuhan Boster Biological Technology, Ltd.) for 40 min to starve the cells. Subsequently, 10 ml 2-DG (10 mM) was added to each well. Following culture for 20 min, the rate of glucose uptake was determined. To determine the lactate production and ATP synthesis of each group, Lactate assay kit II (Invitrogen; Thermo Fisher Scientific, Inc.) and ATP assay kit (Invitrogen; Thermo Fisher Scientific, Inc.), respectively, were used according to the manufacturer's instructions. A total of 2×10⁶ MG63 and Saos-2 cells were lysed in 100 µl of the corresponding assay buffer and homogenized. Following centrifugation, the soluble fractions of lactate and ATP were assayed using 530 and 636 nm wavelengths in an ultraviolet spectrophotometer, respectively.