2.5. Agilent Seahorse XF Cell Mito Stress Test Assay

A total of 6000 cells of each cell line were plated in 80 μL complete media into individual wells of a XF96 cell culture microplate excluding cells from 4 corner control wells (5 technical replicates per assay). The following day, cells were treated with test compounds diluted in complete media followed by incubation for indicated time. Post-incubation cells were washed 2× with warm, sterile filtered Seahorse XF RPMI media supplemented with 1 mM pyruvate, 2 mM glutamine, and 10 mM glucose at pH 7.4. 80 μL of supplemented Seahorse XF RPMI assay media was added back to each well. Cells were allowed to equilibrate for 1 h in a 37 °C CO₂ free humidified incubator before loading into a XFe96 extracellular flux analyzer temperature adjusted to 37 °C (Seahorse Bioscience, Billerica, MA, USA). Sensor cartridges were equilibrated with XF calibrant for 24 h before loading with inhibitors. Inhibitor concentrations were titrated to determine optimal drug concentrations to establish bioenergetic profiles (data not shown), final well concentrations used were 1.0 μM oligomycin, 1.0 μM carbonyl cyanide-4 (trifluoromethoxy) phenylhydrazone (FCCP), and 0.5 μM rotenone and antimycin A. Oxygen and proton concentrations were measured every 8.5 min for 1 h and 35 min, inhibitors (oligomycin, FCCP, rotenone/antimycin A, respectively) to measure mitochondrial bioenergetics were added to the plates through the microinjection ports at the indicated time points. Oxygen consumption rates (OCR) and spare reserve capacity (difference between maximal and basal OCR) are shown. Cells were imaged using the Lionheart FX Automated Microscope (BioTek Instruments, Winooski, VT, USA) to confirm cell adherence before and after assay.