Xf24 flux analyzer

Mitochondrial function was assessed using the XF24 Flux analyzer (Agilent). Briefly, oxygen consumption rates (OCR) and extracellular acidification rates (ECAR) were assessed using freshly isolated mouse BAT, iWAT and eWAT. The tissue was rinsed with non-buffered KHB media containing 111 mM NaCl, 4.7 mM KCl, 2 mM MgSO4, 1.2 mM Na2HPO4, 0.5 mM carnitine and 2.5 mM glucose; they were then cut into pieces (~10 μg) and washed extensively. Then, each piece was placed into a single well of an XF24 Islet plate (Agilent) and covered with a customized screen that allowed free perfusion while minimizing tissue movement. KHB buffer was added to each well, and OCR was measured. For BAT, basal respiration was first measured and then pyruvate (5 μM) was added as substrate. For WAT, basal respiration was recorded before oligomycin (10 μM) was added to inhibit ATP synthesis. FCCP (20 μM) was then supplied to artificially uncouple the mitochondria. Rotenone (3 μM) and Antimycin A (12 μM) were added simultaneously to inhibit complexes I and III of the electron transport chain respectively.

Mitochondrial bioenergetics were measured with the XF24 Flux Analyzer (Agilent Technologies Inc, Santa Clara, CA) as previously described[32 (link);40 (link)]. For measurement of axonal mitochondrial bioenergetics, 10 mm sections of tibial nerves were freshly isolated and placed into the islet capture XF24 microplate (Seahorse Bioscience, North Billerica, MA) in XF media supplemented with 5 mM glucose, 0.5 mM sodium pyruvate, and 1 mM glutamine[32 (link)]. Oxygen consumption rate (OCR) and extracellular acidification rate (ECAR) were measured and normalized to total protein contents of each individual nerve, and were used as indicators of mitochondrial bioenergetic status.
DRG neurons were isolated as described previously[40 (link);43 (link)] and plated in XF24 microplate and cultured overnight in Ham’s F-10 media (Corning Inc., Corning, NY) supplemented with N2 supplement (Thermo Fisher Scientific, Waltham, MA). On the day of assay, media was changed to XF media supplemented with 5 mM glucose, 0.5 mM sodium pyruvate, and 1 mM glutamine for OCR measurement. Carbonyl cyanide-p-trifluoromethoxyphenylhydrazone (FCCP;4 μM) and Rotenone and Antimycin A (2 μM each) (Sigma-Aldrich) were used to determine mitochondrial respiratory properties. The results were normalized to the protein contents of each well.

OCR was measured using the XF24 Flux Analyzer (Seahorse Bioscience) as described (Teperino et al., 2012 (link)). For details see Extended Experimental Procedures.