Agilent Seahorse XF Cell Mito Stress test and oxygen consumption rate

The day before the assay, the Seahorse XF sensor cartridge was hydrated with water and kept in a non-CO₂ humidified incubator at 37 °C. Seahorse XF Calibrant solution was also kept in a non-CO₂ incubator at 37 °C.

On the day of analysis, both young and aged BM-MSCs at the same passage were harvested, counted, and an equal number of cells (7500/well) was seeded in the XF 96-well culture plate. The four corners of the culture plate were left unseeded for background correction. The seeded cells were allowed to adhere to the wells for 1 h at room temperature, and then cultured for 2 h in a humidified incubator at 37 °C under 5% CO₂ in air before undergoing PBM treatment_. After the treatment, the XF 96-well culture plate was returned to the incubator and kept there until the assay’s start. Before the analysis, the culture medium was removed and the cells were washed with pre-warmed XF assay medium consisting of 10 mM glucose, 1 mM sodium pyruvate, and 2 mM glutamine (pH 7.4). For pre-equilibration, the cells were maintained in the assay medium at 37 °C in a non-CO₂ incubator for 1 h. Meanwhile, oligomycin (1.5 μM), carbonyl-cyanide-4-(trifluoromethoxy) phenylhydrazone (FCCP, 1 μM), and rotenone/antimycin A (0.5 μM) compounds from the Seahorse XF Cell Mito Stress Test kit were prepared according to manufacturer’s instructions and loaded into the injection ports of the sensor cartridge in the order of injection.

Measurement of the oxygen consumption rate (OCR, pmol/min), an indicator of mitochondrial respiration, was performed using an XFe96 Extracellular Flux analyzer³⁸. After the calibration of the sensor cartridge in the analyzer, the OCR was measured at baseline, as well as upon consecutive injections of oligomycin, FCCP, and rotenone/antimycin A. After the assay, the cells were washed with phosphate buffered saline (PBS) and lysed with radioimmunoprecipitation assay (RIPA) buffer containing 150 mM NaCl, 1% NP-40, 0.5% sodium deoxycholate, 0.1% SDS, and 50 mM Tris–HCl (pH 8.0) supplemented with 1 × protease inhibitors. Protein concentration of each well was determined using the BCA protein assay. The data was normalized to μg of protein. The Wave software (version 2.6.1) was used to analyze the data.