Xfe24 analyser

Monocytes were adhered to Seahorse plates as described above. Anti-MPO, anti-PR3 or isotype antibodies were added to port A of an XFe24 FluxPak. For ECAR measurements, D-glucose was added to port B. For OCR measurements rotenone or vehicle was added to port B. The Cell plate was added to the Seahorse XFe24 analyser. Six initial basal measurements were performed followed by injection of port A. For ECAR experiments three more measurements were performed before injection of glucose followed by nine further measurements. For OCR experiments one measurement was performed before rotenone injection followed by nine further measurements.

Mitochondria were extracted from the brain tissues using the Mitochondria Extraction Kit (C3606, Beyotime) according to the manufacturer's instructions. Freshly isolated mitochondria (5 μg) were added to Seahorse probe plates (hydrated 6 h before the experiment), and 50 μL of mitochondrial analysis solution (70-mM sucrose, 220-mM mannitol, 2-mM HEPES, 10-mM potassium dihydrogen phosphate, 5-mM magnesium chloride, 1-mM ethylene glycol ditetra-acetic acid, and 0.2% w/v BSA without fatty acid) supplemented with 10-mM succinate or 2-nM glutamate was added to the plates. The samples were centrifuged at 4°C for 20 min at 2000 g. Adenosine diphosphate (ADP), oligomycin (oligo), carbonyl cyanide p-trifluoromethoxy-phenylhydrazone (FCCP), and antimycin A (AA) were added to probe plates A, B, C, and D at the final concentration of 4 mM, 2.5 μg/mL, 4μM and 4μM, respectively. The detection time and cycle times were set, and the probe plates were placed in the Seahorse XFE24 analyser. After centrifugation, the plates were removed, and 450 μL of the corresponding MAS detection solution containing different substrates was added to each well and incubated in a carbon dioxide-free incubator at 37°C for 10 min. After probe plate correction, the oxygen consumption rate (OCR) (pmol/min) of mitochondria was evaluated.