L lactate assay kit

Cells were seeded at approximately 3 × 10⁵ cells/mL in 6-well plates and treated with 2-DG (2 mM) alone for 24 h and buforin IIb alone (2 μM) or both for 12 h. After centrifugation, the culture medium and cells were collected to estimate the L-lactate and ATP content using the ATP content assay kit (Solarbio, Beijing, China) and L-lactate assay kit (Jiancheng Bioengineering, Nanjing, China) according to the manufacturer’s protocol. Lactate dehydrogenase catalyzes the conversion of lactate to pyruvic acid, and the absorbance at 530 nm was determined spectrophotometrically after the addition of a color developing agent, which showed a linear relationship with lactate content. The collected cells were subjected to ultrasonic disruption and centrifuged at 8000× g for 10 min at 4 °C to collect the supernatant for intracellular ATP detection. Creatine kinase catalyzes the production of phosphocreatine from creatine and ATP. The level of ATP was determined by detecting the phosphocreatine content though the colorimetric method of phosphomolybdic acid at 700 nm. Six replicates were performed for each group, and the experiments were repeated three times to confirm the results.

For lactate measurement in serum, the whole blood of treated mice was firstly collected and centrifuged at 4000 rpm for 10 min to obtain the serum. For the analysis of lactate in cell cultures, cells were seeded at approximately 3 × 10⁵ in 6-well plates and treated with different concentrations of ASP and/or AMP for 3 days. After centrifugation, the culture medium and cells were collected. The lactate content was estimated using the l-lactate assay kit (Jiancheng Bioengineering, Nanjing, China) according to the manufacturer’s protocol. Lactate dehydrogenase catalyzed the conversion of lactate to pyruvic acid, and the absorbance at 530 nm was determined spectrophotometrically after the addition of a color developing agent, which showed a linear relationship with lactate content. Three replicates were performed, and the experiments were repeated three times for accuracy.