Cyanide 3 chlorophenylhydrazone cccp

The XF24 biosensor cartridge was activated with 1 ml of XF24 calibrant solution (Seahorse Bioscience, Billerica, MA) per well for 24 h at 37°C in a non-CO2 incubation system. SIRT3 WT and KO BMDMs infected with Mabc for 18 h were seeded at 2 × 10⁴ cells per well and incubated for 24 h at 37°C. The cell plate was incubated for 1 h at 37°C in a non-CO₂ incubation system after the addition of 590 µl assay media in each well. ATPase inhibitor oligomycin A (20 µg/mL, Sigma-Aldrich, MO, USA), uncoupler carbonyl cyanide 3-chlorophenylhydrazone (CCCP, 50 µM, Sigma-Aldrich, MO, USA), and mitochondrial complex I inhibitor rotenone (20 μM, Sigma-Aldrich, MO, USA) were sequentially added to each well after measurement of basal OCR. Seahorse Bioscience XF24 analyzer (Seahorse Bioscience, Billerica, MA) was used to measure the oxygen consumption rate of the entire process.

BV-2 cells (3 × 10³/well) were incubated in media containing the vehicle, LPS NC or siATF5. Firstly, basal OCR was measured. Subsequently, ATPase inhibitor oligomycin A (20 µg/mL, Sigma-Aldrich, Louis, MO, USA), uncoupler carbonyl cyanide 3-chlorophenylhydrazone (CCCP, 50 µM, Sigma-Aldrich, Louis, MO, USA), and mitochondrial complex I inhibitor rotenone (20 µM, Sigma-Aldrich, Louis, MO, USA) were added and measured at 37 °C using an XF24 analyzer (Seahorse, North Billerica, MA, USA).