Xfp cell culture microplate

Oligomycin was purchased from EMD Millipore, and FCCP and rotenone were purchased from Sigma. C2C12 cells were seeded at 30,000 cells/well, respectively in an XFp cell culture microplate (Seahorse Bioscience) and analyzed the following day. On the day of analysis, the cells were washed once with Seahorse respiration buffer (8.3 g/l DMEM, 1.8 g/l NaCl, 1 mM pyruvate, 20 mM glucose, pen/strep), placed in 0.5 ml Seahorse respiration buffer, and incubated in a CO₂-free incubator for 1 hr. Port injection solutions were prepared as follows (final concentrations in assay in parentheses): 10 μM Oligomycin (1 μM final), 500 μM C20:4-Gly (50 μM final), and 30 μM rotenone (3 μM final). The Seahorse program was run as follows: basal measurement, 3 cycles; inject port A (Oligomycin), 3 cycles; inject port B (compounds), 3 cycles; inject port C (rotenone), 3 cycles. Each cycle consisted of mix 4 min, wait 0 min, and measure 2 min. For data expressed as “pre” and “post,” the average of the three measurements following Oligomycin injection were averaged for the “pre” state and the average of the three measurements following N-acyl amino acid injection were averaged for the “post” state. Experiments using BSA included fatty acid free BSA in the port injection solution (e.g., 500 μM C20:4-Gly with 40 mg/ml BSA).