4.15. Seahorse analysis of mitochondrial function

Measures of oxygen consumption was determined with a Seahorse XFe96 Analyzer (Agilent). General mitochondrial function was assessed according to the Seahorse XF Cell Mito Stress Kit protocol (Agilent). Briefly, cells were seeded at 40,000 per well in quadruplicate on an XFe96 microplate and allowed to settle down overnight. Immediately before the assay, cells were supplemented with 10 mM glucose and 1 mM glutamine and then sequentially challenged with 1 μM oligomycin, 1 μM of FCCP, and 1 μM each of antimycin A and rotenone. We assessed the individual ETC complex activity according to an established protocol [63]. Briefly, 40,000 cells were plated in quadruplicate on an XFe96 microplate and allowed to seed overnight. Immediately before assay, cells were overlaid with 175 μL of mitochondrial assay solution [33] supplemented with the Seahorse Plasma Membrane Permeabilizer (Agilent), 4 mM ADP (Sigma- Aldrich), and 10 mM sodium pyruvate (Sigma-Aldrich) with 1 mM malate (Sigma-Aldrich). Cells were then sequentially subjected to 2 μM rotenone (Sigma-Aldrich), 10 mM succinate (Sigma-Aldrich), 2 μM antimycin A (Sigma-Aldrich), and 10 mM ascorbate (Sigma- Aldrich) with 100 μM N,N,N′,N′-tetramethyl-ρ-phenylene diamine (Sigma-Aldrich). Glycolytic and mitochondrial ATP production rates were determined according to the XF Real-Time ATP Rate Assay Kit protocol (Agilent). Briefly, 40,000 cells were seeded overnight on an XFe96 microplate in prepared medium mimicking Seahorse XF RPMI, pH 7.4 supplemented with 10 mM glucose (Sigma-Aldrich) and 2 mM glutamine (VWR). Cells were then overlaid with fresh medium and sequentially subjected to 1 μM oligomycin and then concomitant 1 μM rotenone and antimycin A. Glycolytic and mitochondrial ATP production rates were calculated according to the “Quantifying Cellular ATP Production Rate Using Agilent Seahorse XF Technology” White Paper (Agilent).