Mito stress test kit

The metabolic status was investigated on an XFe96 Extracellular Flux analyzer (Seahorse Bioscience) with standard 96-well Seahorse microplates (Agilent, 101,085–004). A Mito Stress test kit (Agilent, 103,015) was applied, and the oxygen consumption ratio (OCR) and the extracellular acidification rate (ECAR) were measured. In brief, VCaP, C4-2B or RWPE-1 cells were seeded into 96 well plates with or without (R)-9b for 12 h and allowed to form a monolayer. Thereafter, medium was then replaced by 180 μl of non-buffered Dulbecco’s modified Eagle’s medium (DMEM) containing 10 mM glucose, 2 mM glutamine and 1 mM pyruvate. The cells were incubated in a CO₂-free incubator at 37°C for 1 h to allow for temperature and pH equilibration before being loaded into the XFe96 analyzer. The injection sequence was programmed as: 1st, oligomycin (1 μM at final concentration); 2nd, FCCP (1 μM at final concentration); 3rd, rotenone and antimycin A (0.5 μM and 0.5 μM at final concentrations, respectively). ATP-linked OCR was measured by subtracting the OCR at oligomycin addition from baseline cellular OCR. The data was normalized with cell number and analyzed using software Wave (version 2.2.0, Seahorse Bioscience) for visual presence.

SK-N-SH cells transfected with SSOs for 24 h were trypsinized and seeded in a Seahorse XF96 cell culture microplate (2 × 10⁴ cells/well). After incubation for 24 h, the medium was replaced with Seahorse XF medium and incubated for 1 h at 37 °C in a non-CO₂ incubator. The oxygen consumption rate (OCR) was measured using a Seahorse XFe96 Analyzer (Agilent, CA, USA) with the Mito Stress Test Kit according to the manufacturer's instructions. The measurements were normalized to the cell numbers.