Bcaa kit

SM injection is a TCM extracted from Panax ginseng (Panax ginseng C.A. Mey, steamed and dry) and Ophiopogon japonicus
[Ophiopogon japonicus (L.f.) Ker-Gawl, root]. SM injection is mass produced as a patented drug based on the national standards approved by the CFDA from Chia Tai Qingchunbao Pharmaceutical Co., Ltd. (Hangzhou, China). TMZ was purchased from Servier Industry Laboratories (France). The Assay kits used to measure the glucose, FFA, LA, and PA contents were purchased from Solarbio Science & Technology Co., Ltd. (Beijing, China). The BCAA kit was obtained from Sigma-Aldrich Co., LLC (USA). Acetonitrile of high-performance liquid chromatography (HPLC) grade was obtained from Fisher Scientific (New Jersey, USA), and purified water was purchased from Robust Company (Hangzhou, China).

BCAA concentration was detected using the BCAA kit (Sigma, MAK003) according to the manufacturer’s instructions. The cells were seeded in the six-well plates for 24 h, the cell numbers were counted, collected and be rapidly homogenized in 100 μL of cold BCAA Assay buffer for detecting BCAA. Concentration of BCAA is calculated by the formulation: C = S_a/S_v (S_a = Amount of BCAA in unknown sample (nmole) from standard curve, S_v = Sample volume (mL) used). For determining glucose and lactate, Huh7 and HepG2 cells were seeded in the six-well plates. After 24 h, The cell culture medium was collected. Glucose and lactate were determined using the glucose assay kit (Sigma, MAK181) and lactate assay kit (Sigma, MAK064) respectively according the manufacturer’s instructions. Glucose consumption was determined by subtracting the final medium glucose concentration from the initial medium glucose concentration in the culture medium.

BCAA concentration in plasma and supernatant of cultured cells were determined using a BCAA kit (Sigma, MAK003) according to the manufacturer’s instructions.