Xf plasma membrane permeabilizer reagent

Mouse C2C12 cells were transfected twice at 0 and 72 h, with 100 nm siRNA of negative control, METTL20, or ETFβ. The OCR of transfected cells was measured in an XF96 extracellular flux analyzer (Seahorse Biosciences, MA) at 120 h. The assay medium consisted of 70 mm sucrose, 220 mm mannitol, 10 mm KH₂PO₄, 5 mm MgCl₂, 2 mm HEPES, 1 mm EGTA, 0.2% (w/v) fatty acid-free BSA (pH 7.4), and 4 mm ADP. The cells were treated with 1 nm XF plasma membrane permeabilizer reagent (Seahorse Biosciences). When the OCR reached a stable baseline, 40 μm palmitoyl-l-carnitine chloride supplemented with 0.2 mm malate was added. Respiration was normalized to cell number by the sulforhodamine B colorimetric assay (48 (link)). p values for the OCR were calculated with a paired Student's t test.

Measurement of oxygen consumption was performed using a Seahorse XF^e24 Flux Analyzer (Seahorse Biosciences). 30’000 cells were seeded in XF24 cell culture microplates and grown overnight in DMEM containing 10% FBS, 2mM L-Glutamine, 25mM Glucose, and Penicillin/Streptomycin. Experiments on primary or immortalized, permeabilized MEFs were carried out at 37°C in Mitochondrial Assay Solution (MAS, containing 70 mM sucrose, 220 mM mannitol, 10 mM KH₂PO₄, 5 mM MgCl₂, 2 mM Hepes, 1 mM EGTA, 0.2% fatty acid free BSA; pH 7.2). Cells were permeabilized with 1 nM XF Plasma Membrane Permeabilizer reagent (Seahorse Bioscience) and provided with 5 mM pyruvate, 0.5 mM malate, 2 mM dichloroacetate and 1 μM oligomycin one hour before the assay. Basal oxygen consumption was measured before injection. At the times indicated, the following compounds were injected: fCCP (final concentration 0.4 μM (primary MEFs) or 2 μM (immortalized MEFs)), succinate/rotenone (10 mM/1 μM), antimycin A (1 μM). Each measurement loop consisted in 30 sec mixing, 1 min waiting, and 2 min measuring oxygen consumption. For immortalized cells, OCR data were corrected for cell number by nuclear staining with DAPI.

Measurement of oxygen consumption was performed using a Seahorse XF^e24 Flux Analyzer (Seahorse Biosciences). 35,000 cells were seeded into XF24 cell culture microplates and grown overnight in DMEM containing 10% FBS, 2 mml-glutamine, 25 mm glucose, and penicillin/streptomycin. Experiments were carried out at 37 °C in Mitochondrial Assay Solution (containing 70 mm sucrose, 220 mm mannitol, 10 mm KH₂PO₄, 5 mm MgCl₂, 2 mm Hepes, 1 mm EGTA, and 0.2% fatty acid-free BSA, pH 7.2). Cells were permeabilized with 1 nm XF Plasma Membrane Permeabilizer reagent (Seahorse Bioscience) and provided with 5 mm pyruvate, 0.5 mm malate, 2 mm dichloroacetate, and 1 μm oligomycin for 1 h before the assay. Basal oxygen consumption was measured before further treatment. At the times indicated, the following compounds were injected: carbonyl cyanide-4-(trifluoromethoxy)phenylhydrazone (final concentration, 2 μm), succinate/rotenone (10 mm/1 μm), and antimycin A (1 μm). Each measurement loop consisted of 30 s mixing, 1 min waiting, and 2 min measuring oxygen consumption.