Mitochondrial Respiration Profiling in Cells

Mitochondrial function was determined in intact cells by real-time respirometry (Seahorse XFe24; Agilent). MDA-MB-231 and EO771 cells were seeded at 1.0 × 10⁴ cells/well in an XFe 24-well plate (Agilent). Once cells reached 80% confluency, they were treated with a vehicle (0.01% DMSO) or varying concentrations of BAM15 for 16 h. Following treatment, media was removed, and cells were incubated at 37°C for 1 h in XF DMEM medium (pH 7.4) supplemented with 1 mM pyruvate and 2 mM glutamine without CO₂. Cells were then serially injected with 10 mM glucose, 1 μM oligomycin, 1 μM FCCP, and 0.5 μM rotenone and antimycin A. Components of mitochondrial function were calculated as described previously [67 (link)]. Data were normalized to nuclear content by staining live cells after assay with 20 μM Hoechst 33342 (ThermoFisher Scientific) and reading fluorescence on an automated microplate reader (Biotek) at excitation/emission 350/461 nm. Mitochondrial respiration was quantified as described previously [18 (link)].