Enzychrom lactate assay kit

Glucose consumption, lactate level, and intracellular ATP levels were detected using a commercially available Glucose Assay Kit (Sigma-Aldrich, Seoul, Korea), the EnzyChrom™ Lactate Assay Kit (Bioassay Systems, Hayward, CA, USA), and EnzyLight^TM ATP assay kit (Bioassays Systems, Hayward, CA, USA), respectively [18 (link)].

Glucose consumption and the lactate level were calculated using the Glucose (HK) Assay Kit (Sigma-Aldrich) and the EnzyChrom™ Lactate Assay Kit (Bioassay systems). Intracellular ATP levels were measured using a commercially available EnzyLight^TM ATP assay kit (Bioassays Systems, CA). The ATP levels of the treated cells were normalized to the ATP levels of the untreated cells. The rate of oxygen consumption and the extracellular acidification rate were evaluated using a real time Seahorse XF24 Analyzer (Seahorse Bioscience, North Billerica, MA, USA) as previously described58 (link). Briefly, 2 × 10⁴ cancer cells were seeded at 24 h before the 2-DG and plasma treatment. Before beginning the measurements, the cells were placed in a phenol red free DMEM medium (supplemented with 25 mM glucose, 2 mM glutamine, and 1 mM sodium pyruvate, and without serum) and pre-incubated for 1 h at 37°C in atmospheric CO₂. To determine the OCR values, these values were normalized for the protein content of each sample.

Venous blood samples were collected by venipuncture at the indicated time points (Figure 1). Blood samples were collected into ethylenediaminetetraacetic acid (EDTA)-containing tubes. The EDTA-containing tubes for plasma separation were immediately centrifuged at 3000 rpm (approximately 1700× g) for 10 min at 4 °C. Plasma samples were stored at −80 °C until analysis. Plasma total protein, albumin, albumin/globin ratio (A/G), total bilirubin, aspartate aminotransferase (AST), alanine aminotransferase (ALT), lactate dehydrogenase (LDH), γ-glutamyl transpeptidase (γ-GTP), creatine kinase (CK), amylase, total cholesterol (T-CHO), low-density lipoprotein cholesterol (LDL-CHO), high-density lipoprotein cholesterol (HDL-CHO), aldolase, triglyceride, free fatty acid, uric acid, blood urea nitrogen, creatinine, glucose, bile acid, blood concentration of calcium ion, chloride ion, potassium ion, sodium ion, and inorganic phosphorus were measured by Koutou-Biken Co. (Tsukuba, Japan). Glycerol, lactate, and ketone body concentrations were measured with an EnzyChrom™ Glycerol Assay Kit, EnzyChrom™ Lactate Assay Kit, and EnzyChrom™ Ketone Body Assay Kit (Bioassay systems, Hayward, CA, USA).