Picoprobe acetyl coa fluorometric assay kit

Acetyl-CoA was measured using the PicoProbe™ Acetyl-CoA fluorometric assay kit (BioVision K317-100). HDAC activity was measured using the HDAC activity colorimetric assay kit (BioVision K331-100). Specifically, 1.5 million cells were lysed and diluted to 85μl in ddH₂O in a 96-well plate. 10μl of 10×HDAC assay buffer and 5μl of the HDAC colorimetric substrate were added and mixed thoroughly. The plate was incubated at 37^oC for 2h before adding 10μl of lysine developer. The plate was incubated at 37^oC for 30min and measured at 405nm. HDAC activity was expressed as the relative O.D. value per mg of protein.

Immunoprecipitated full-length Flag–KAT2A from 293 cells was immobilized on anti-Flag-agarose beads and incubated with purified histone H3 (4 μM) in HAT buffer in the presence of 0.009 μM, 0.027 μM, 0.082 μM, 0.247 μM, 0.741 μM, 2.222 μM, 6.667 μM, or 20.000 μM of acetyl-CoA or succinyl-CoA at 25 °C for 5 min. The reaction mixture was precipitated via centrifugation at 1,000g for 5 min. The production of CoA in the supernatant was quantitatively analysed using a modified protocol from the PicoProbe Acetyl-CoA Fluorometric Assay Kit (BioVision). In brief, 25 μl supernatant was mixed with 1 μl PicoProbe, 1 μl acetyl-CoA substrate mix, 2.5 μl acetyl-CoA enzyme mix, and 20.5 μl acetyl-CoA assay buffer. The CoA can react to form NADH, which interacts with OicoProbe to generate fluorescence (E_x/E_m = 535/587 nm). An identical reaction was set up for each acetyl-CoA or succinyl-CoA titration without KAT2A protein and used as the blank control. Data are presented as the means ± s.d. of three independent experiments.

Free fatty acid levels in WT and Nox4^−/− BMDMs or peritoneal macrophages were measured using the Free Fatty Acid Quantification Colorimetric/Fluorometric Kit (K612-100, Biovision) according to the manufacturer’s instructions. Intracellular acetyl-CoA levels in WT and Nox4^−/− BMDMs or peritoneal macrophages were measured by PicoProbe™ Acetyl-CoA Fluorometric Assay Kit (K317-100, Biovision) according to the manufacturer’s instructions.

Quantification of intracellular acetyl-CoA was performed using the PicoProbe Acetyl-CoA Fluorometric Assay Kit (catalog no. K317–100, BioVision, USA) according to the manufacturer’s instructions. hPTCs were incubated for 48 hours with 100 nM CsA, 100 nM cisplatin, and vehicle for control. Cells were lysed on ice, and fluorescence was measured with a plate reader (PerkinElmer, USA).