Bcaa detection kit

The human plasma BCAA and BCKA were measured by Hangzhou Calibra Diagnostics (China) with isotope dilution LC-MS as previously described [20 (link)]. Standard l-leucine, l-isoleucine, l-valine, α-ketoisovaleric acid, α-keto-β-methylvaleric acid, α-ketoisocaproic acid, L-¹³C₁-leucine and salicylic acid were purchased from Sigma-Aldrich. In brief, the plasma samples were pretreated with protein precipitation. LC-MS was performed with an Agilent Zorbax SB-C18 column and Sciex TripleQuad 4500MD. The animal plasma BCAA and cellular BCAA were measured with a BCAA detection kit (Biovision, USA) according to the manufacturer's instructions. The animal plasma BCKA and cellular BCKA were determined by HPLC as previously described [21 (link)].

ADSCs (2 × 10⁶ in quantity) transfected with shNC or shBcat1#1 were used for intracellular metabolite concentration measurement. Each metabolite level was determined using the following assay kits: BCAA detection kit (BioVision, K564‐100, α‐ketoglutarate assay kit (Sigma‐Aldrich, MAK054), glutamate assay kit (Sigma‐Aldrich, MAK004), citrate assay kit (Sigma‐Aldrich, MAK057), fumarate assay kit (Sigma‐Aldrich, MAK060), succinate assay kit (Sigma‐Aldrich, MAK184), and malate assay kit (Sigma‐Aldrich, MAK196). BCKA concentration was measured by high‐performance liquid chromatography (HPLC) as previously described.^[42] Intracellular metabolite levels were normalized to the ADSCs transfected with shNC.