Xf cell mito stress test assay

OCR was measured using XF 96 Extracellular Analyzer instrument (Seahorse Bioscience Inc., Chicopee, MA). MCF7 and BT474 cells were plated at 30,000 cells per well on XF96 cell culture microplate in 100 μl of culture media. Cells were treated with 10 μM 8-Cl-Ado for 18 hrs. Media was then replaced with fresh XF assay medium (Seahorse Bioscience Inc.), supplemented with 17.5 mM Glucose and 2 mM Sodium Pyruvate, (175 μl/well) and is incubated in a CO₂ free chamber of XF Prep station for 1 hr. XF Cell Mito Stress Test assay was performed as per the manufacturer’s instructions using the following final concentrations; 1.25 μM oligomycin, 1 μM FCCP, 0.75 μM antimycin and 1.25 μM rotenone (Seahorse Bioscience Inc.). Each assay was repeated at least twice. For ECAR, the culture media was replaced with glycolysis stress test media (prepared in accordance with Seahorse Glycolysis stress kit) supplemented with 2 mM of fresh L-Glutamine. For the glycolysis assay, all ports were injected with 25 μl of drugs for the following final concentrations; Port A-10 mM glucose, Port B −1.25 oligomycin, Port C– 100 mM 2-deoxyglucose.
Perchloric acid was used to extract NTPs from MCF-7 and BT-474 cells treated with 10 μM 8-Cl-Ado and neutralized extracts were analyzed by HPLC (Waters 600E System Controller; Waters Corp., Milford, MA, USA) as described [2 (link)].