Glucose assay kit

The level of glucose and lactate in supernatant of MELF were determined by using the glucose assay kit (Applygen, Beijing, #E1011) the lactate assay kit (St. Louis, MO, USA, #MAK064) according to the manufacturer’s instructions, respectively. The lactate dehydrogenase (LDH) in supernatant of MELF released by damaged cells was measured by LDH assay kit (Beyotime, China, A020-1) according to the instructions.

To detect glucose uptake, lactate production, lactate dehydrogenase activity, ATP level, and hexokinase activity in gastric cancer cells, the Glucose Assay Kit (Applygen, Beijing, China), Lactate Acid Assay Kit (Jiancheng Bioengineering Institute, Nanjing, China), Lactate Dehydrogenase Activity Assay Kit (Jiancheng bioengineering institute), luciferase-based ATP Assay Kit (Beyotime, Shanghai, China), and Hexokinase Activity Assay Kit (Comin, Suzhou, China) were used according to the manufacturer’s protocols. All values were normalized to cell number or total protein levels.

The glucose content of the medium treated as described above was measured using a glucose assay kit (Applygen Technologies Inc.). The cells were collected and lysed, and the total protein concentration was determined by BCA assay (Applygen Technologies Inc.) to correct for the cell counts.

The cells were resuspended in Fresh IMDM containing 10% FBS and counted by a cell counting plate. Cells (5 × 10⁵/well) were seeded into a 6-well plate, and medium without inoculation was used as the control. After cultured for 24h, the culture medium was collected to measure glucose and lactate concentrations. The glucose concentrations were assayed using the Glucose Assay Kit (E1010, Applygen Technologies Inc.) based on the glucose oxidase method. Optical density (OD) 550 values were measured using Spectra Max M5. Glucose concentration (mmol/L) = concentration standard (mmol/L) × (OD_sample- OD_blank)/(OD_{standard product}- OD_blank). Glucose consumption was calculated by deducting the measured glucose concentration in the medium of the experimental groups from the glucose concentration of the control. Lactate production was determined by using the Lactate Assay Kit (A019-2-1; Nanjing Jiancheng Bioengineering Institute) according to the manufacturer’s instructions. Optical density (OD) 530 values were measured using Spectra Max M5. Lactate concentration (mmol/L) = concentration standard (mmol/L) × (OD_sample- OD_blank)/(OD_{standard product}- OD_blank). PKM2 inhibitor (HY-103617, Medchemexpress).

MCF7 cells were seeded on 24-well plates. The cells were washed twice with culture medium, and fresh culture medium was added. Twelve hours later, the cell culture medium was collected and frozen at −80 °C, and the number of cells in each well was counted using a cell counter (Invitrogen). The glucose concentration in the collected cell culture medium were determined by using the glucose assay kit (Applygen, Beijing, China), the lactate concentration were determined by the lactate assay kit (Kinghawk, Beijing, China) according to the manufacturer’s protocol.

Briefly, 2 × 105 A549/DDP cells were seeded into 6-well plates and cultured for 24 h. The culture medium and cells were collected separately, and glucose concentration and lactate production were measured using the Glucose assay kit (Applygen, Beijing, China) and the Lactic Acid assay (Jiancheng Bioengineering Institute, Nanjing, China) in accordance with the manufacturers' protocols, and the glucose consumption level or the lactate production level was divided by the number of cells.