Acetyl coa assay kit

E. coli cells were cultured in M9 medium until early mid-log phase (OD₆₀₀, 0.8 to 1.0) and collected for the quantitative determination of intracellular ATP/ADP ratios and acetyl-CoA levels using the ATP assay kit (Solarbio, Beijing, China), ADP assay kit (Solarbio, Beijing, China), and acetyl-CoA assay kit (Solarbio, Beijing, China). Extraction and quantification for intracellular ATP/ADP ratios were carried out according to the ATP and ADP assay kit instructions, and the analysis of ATP/ADP ratios was performed with an Agilent 1260 series HPLC system. The analytical column was a Thermo 250-mm by 4.0-mm ODS-2HYPERSIL C₁₈ column. The intracellular acetyl-CoA level was analyzed according to previous publications (52 (link), 53 (link)).

Cells were seeded into six-well plates at a density of 1 × 10⁶ cells in 2 ml RPMI 1640 medium per well and cultured overnight. Cells were then treated with or without 10 μM KU60019 and maintained at 1% O₂ for 10–12 h. Glucose consumption, lactate production, ATP production, citrate production, succinate production, fumarate production, and PFKP and CS activity were detected using glucose assay kit (Solarbio® BC2500), LA assay kit (Solarbio® BC2230), ATP content assay kit (Solarbio® BC0300), citric acid (CA) content assay (Solarbio® BC2150), micro mitochondrial citric acid content assay kit (Solarbio® BC2175), succinate colorimetric assay kit (Sigma® MAK184), pyruvate (PA) assay kit (Solarbio® BC2200), acetyl-CoA assay kit (Solarbio® BC0980), fumarate assay kit (Sigma® MAK060), PFKP test kit (Nanjing Jiancheng Bioengineering Institute A129), and citroyl synthetase kit (Nanjing Jiancheng Bioengineering Institute A108) according to instruction of the manufacturers, respectively. All experiments were performed at least three times and the data were normalized by the cell numbers or protein content.

A mitochondria isolation kit (ab110170, Abcam) was used in this experiment^{16 (link),17 (link)}. In brief, cultured NSCs were frozen and thawed to weaken the membranes and suspended in Reagent A. Then, the cells were homogenized and centrifuged at 1000× g at 4 °C for 10 min. The supernatant was collected (SN1), and the pellet was resuspended with Reagent B. After homogenization and centrifugation, the supernatant was collected again (SN2). SN1 and SN2 were mixed thoroughly and centrifuged at 12,000× g for 15 min at 4 °C. The pellet was collected in Reagent C supplemented with protease inhibitors and used for acetyl-CoA quantification.
Acetyl-CoA quantification was conducted using an acetyl-CoA assay kit (Solarbio life sciences, Beijing, China). Briefly, 5×10⁶ cells or isolated mitochondria were collected and incubated in extraction buffer for 30 min. The cells were subjected to sonication and centrifuged at 8000× g at 4 °C for 10 min. The supernatants were collected and supplemented with acetyl-CoA assay buffer. The 340 nm absorbance values were measured at 20 s (A_20s) and 80 s (A_80s). The difference between A_80S and A_20S was used to calculate the relative level of acetyl-CoA.