Seahorse xfe24 cell culture microplate

Cells were plated in a Seahorse XFe24 Cell Culture Microplate (Agilent). All Seahorse experiments in cells were performed at 24 hours after individual stimuli. At the end of the treatment, cells were washed twice with Agilent Seahorse XF Media (Agilent) supplemented with 1 mM pyruvate, 2 mM L-glutamine, and 2 mM D-glucose; a final volume of 525 μl was placed in each well. Cells were then incubated in a 0% CO₂ chamber at 37°C for 1 hour before being placed into a Seahorse XFe24 Analyzer (Agilent). For oxygen consumption rate (OCR) and extracellular acidification rate (ECAR) experiments, cells were treated with 1 μM oligomycin, 2 μM carbonyl cyanide p-trifluoromethoxy phenylhydrazone (FCCP), and 0.5 μM rotenone/antimycin. A total of three OCR and pH measurements were taken after each compound was administered. All Seahorse experiments were repeated at least three times.

Stable cell lines were seeded at 10,000 cells/well onto a 0.1 % gelatin coated Seahorse XF^e24 cell culture microplate (#100777-004, Agilent Technologies, Santa Clara, CA, USA) and cultured overnight in DMEM (#10-013-CV, Corning; Manassas, VA, USA) supplemented with 10 % FBS and 1 % penicillin/streptomycin. The next day, growth media was replaced with 675 μL/well XF DMEM media, pH 7.4 (#103575-100, Agilent Technologies; Santa Clara, CA, USA) supplemented with 10 mM glucose (#103577-100, Agilent Technologies; Santa Clara, CA, USA) and 10 mM sodium pyruvate (#103578-100, Agilent Technologies; Santa Clara, CA, USA) without phenol red or FBS. Cells were incubated in a CO₂ free incubator for 1 h to degas the media. The oxygen consumption rate (OCR) and extracellular acidification rate (ECAR) were simultaneously measured in an XF^e24 Seahorse Extracellular Flux Analyzer according to manufacturer’s protocol. Mitochondrial stress tests were performed via sequential injection of 1 μM oligomycin, 2 μM trifluoromethoxy carbonylcyanide phenylhydrazone (FCCP), and 1 μM rotenone/antimycin A using the Seahorse XF Cell Mito Stress Test Kit (#103015-100, Agilent Technologies, Santa Clara, CA, USA) following the manufacturer’s protocol. Calculations for the mitochondrial stress test readouts were performed as specified by the manufacturer.

H9c2 cells were plated at 18,000 cells per well on Seahorse XFe24 Cell Culture Microplate (Agilent, Cat#101085-004, Santa Clara, CA) and treated with LPS (1 μg/ml), ΨDrp1 (1μM) and/or PFTμ (5 μM) for 24 hours. At the end of the experiment, cells were washed twice with Agilent Seahorse XF Media, supplemented with 1 mM pyruvate, 2 mM L-glutamine, and 2 mM D-glucose; a final volume of 525 μl was placed in each well. Cells were then incubated in a 0% CO2 chamber at 37°C for 1 h before analysis on a Seahorse XFe24 Analyzer (Agilent, Santa Clara, CA). MitoStress assays were performed by treating the cells sequentially with 1 μM oligomycin, 2 μM carbonyl cyanide p-trifluoromethoxy phenylhydrazone (FCCP), and 0.5 μM rotenone/antimycin. A total of three oxygen consumption rate (OCR) and extracellular acidification rates (ECAR) measurements were taken after each compound was administered. Metabolic balance between aerobic and anerobic metabolism was assessed averaged over each compound as a ratio of OCR/ECAR.