Seahorse Mito stress test assay

All wells of the Seahorse XF^e 96-well plate were treated with poly-D-lysine and then cells (2 × 10⁴ cells/well) were plated and allowed to adhere overnight. The Seahorse XF^e sensor cartridge was hydrated overnight according to manufacturer’s instructions. The next day, the cell culture media in the XF^e 96-well plate was removed and each well was washed once with Seahorse XF Assay Medium. Fresh Assay Medium (180 μL) containing either BenSer (10 mM), BCH (10 mM) or vehicle control (sterile endotoxin-free water; Sigma) was added to each well. The XF^e 96-well plate was then incubated for 1 h at 37 °C in a non-CO₂ incubator, as per the manufacturer’s instructions. The overnight pre-hydrated sensor cartridge was then loaded with the mitochondrial inhibitors oligomycin, FCCP, and rotenone and antimycin A, which were provided in the Mito Stress Test kit and diluted just prior to use according to manufacturer’s instructions. These inhibitors were delivered sequentially from ports A (oligomycin; 1.3 μM), B (FCCP; MCF-7 0.25 μM; HCC1806 and MDA-MB-231 0.5 μM), and C (rotenone 0.5 μM and antimycin A 0.5 μM) in all wells, to measure ATP–linked respiration, maximal respiration, and non-mitochondrial respiration, respectively.

The loaded sensor cartridge was then calibrated in the Seahorse XF^e96 machine according to manufacturer’s instructions, before being loaded into the XF^e 96-well plate for commencement of the Mito Stress Test Assay. Oxygen consumption rate (OCR) and extracellular acidification rate (ECAR) in each well was measured at 6.5 min intervals for 130 min. These measurements captured three baseline measurements (“basal respiration”), four measurements post-oligomycin injection (“ATP-linked respiration”), four measurements post-FCCP injection (“maximal respiration”), and four measurements post-rotenone/antimycin A injection (“non-mitochondrial respiration”). Proton leak and spare respiratory capacity were calculated from the OCR measurements according to manufacturer’s instructions.