Lactic acid assay kit

Transfected A549 and H1299 cells were seeded in 6-well plates (5 × 10⁵) and the culture media were harvested 48 h after transfection. The glucose and lactate levels were measured using a Glucose Assay Kit (Sigma-Aldrich, USA) and a Lactic Acid Assay Kit (Sigma-Aldrich, USA), respectively, according to the manufacturer’s protocol. The values were normalized to the total protein concentration. All experiments were performed in triplicate.

Examination of glucose uptake, lactic acid, and ATP production in HeLa cells was conducted via glucose uptake colorimetric assay kit (Biovision, Milpitas, CA, USA), lactic acid assay kit (Sigma St. Louis, MO, USA), and ATP colorimetric kit (Sigma).

LN229 and U251 cells transfected with indicated plasmids or oligonucleotides were injected into 12-well plates (2 × 10⁴ cells/well), and then cells were incubated in 1.5 mL of Dulbecco’s modified Eagle medium. After 48 h, partial culture medium for each well was collected for assay of glucose consumption and lactate production by glucose assay Kit (Biovision, Milpitas, CA, USA) and lactic acid assay kit (Sigma-Aldrich Chemical Company, Louis, Missouri, USA), respectively. In addition, LN229 and U251 cells were lysed for measurement LDHA enzyme activity, intracellular ATP, and ROS level by Lactate dehydrogenase activity detection kit (Sigma-Aldrich Chemical Company), ATP assay kit (Solarbio, Beijing, China), and reactive oxygen species assay kit (Solarbio), individually. All assay kit was performed in line with the producer’s direction.

The flies (n ≥ 50, 25 days after eclosion) were homogenized thoroughly in cold RIPA buffer (Solarbio, #R0020) supplemented with cocktail protease inhibitor (bimake, #B14001) with tissue homogenizer (Next Advance) and incubated on ice for 30 min. Samples were centrifuged at 13800 g for 10 min at 4°C, and the supernatants were collected for the energy system analysis. The content of ATP and lactate was determined by applying the ATP Assay Kit (Beyotime, #S0026) and Lactic Acid Assay Kit (Sigma‐Aldrich, MAK064), respectively, according to the manufacturer's instructions.

Blood samples of mice were collected using the process described previously [36 (link)] from 0.5 to 6h after metformin was given via oral gavage. Blood lactic acid levels were measured by lactic acid assay kit (Sigma-Aldrich) and metformin concentrations were determined by HPLC, as reported previously [37 (link)].