Acetyl coa assay kit

For the measurement of cytosolic acetyl-CoA, primary peritoneal macrophages were stimulated with different strains or components for 4 h and were lysed with lysis buffer (1% Triton X-100, 20 mM Tris-HCl, pH 7.4, 150 mM NaCl) on ice for 10 min. The lysates were spun at 20,000× g for 10 min at 4 °C, the pellets were discarded, and the supernatants were used for acetyl-CoA measurement with an Acetyl-CoA Assay Kit (Sigma). Freshly isolated primary peritoneal macrophages were rested in RPMI-1640 medium with 2 mM sodium acetate (NaAc) for 24 h at 37 °C in an incubator, and the level of acetyl-CoA was measured.

The concentration of acetyl-CoA was detected by using Acetyl-CoA assay kit (Sigma MAK039) according to manufacturer’s protocols. Briefly, cells were harvested in RIPA buffer (Beyotime Biotech Co. Ltd., China). The cellular protein was precipitated with PCA (BioVision K808–200). The supernatant was neutralized by potassium bicarbonate and then measured by using fluorescence assay method (λ_ex = 535/ λ_em = 587 nm). The concentration of citrate was measured by using citrate colorimetric assay kit (BioVision K655–100) according to manufacturer’s protocols. Briefly, cells were rapidly homogenized with citrate assay buffer, then centrifuged at 15,000 × g for 10 min to remove cell debris. The cellular protein was precipitated with PCA and optical density (OD) value was measured at 570 nm.

Acetyl-CoA was analyzed according to a previously described method [7 (link)]. Yeast colonies of CENP01, CENAA01, and CENPAA01 and wild-type CEN.PK2-1C were pre-cultured in 5-mL CM medium in 50-mL tubes overnight at 30 °C, 225 rpm. The pre-cultures were diluted into fresh 50-mL CM medium to a final OD₆₀₀ of 0.05. Cells were harvested during mid-log phase by centrifugation at 12,000 rpm for 5 min. 10-mL pre-chilled (−80 °C) methanol was added to quench cell metabolism and centrifuged at 12,000 rpm for 5 min to remove the supernatant. 2 mL boiling ethanol was added to cell pellets and the mixture was treated thoroughly by glass beads for 5 min (vortex) to release intracellular metabolites. The supernatant was vacuum dried after centrifugation and re-suspended in 200 μL ddH₂O. The resulting solution containing acetyl-CoA was analyzed by an Acetyl-CoA Assay Kit (Sigma-Aldrich, St. Louis, MO, USA). Acetyl-CoA concentration obtained was an average of biological duplicates, normalized by dry cell weight.

Intracellular levels of acetyl-CoA were examined using an acetyl-CoA assay kit (Sigma-Aldrich, St. Louis, MO, USA).