Xf mito stress test kit

According to the manufacturer’s instructions, OCR was measured with the Seahorse XF 96 Analyzers (Seahorse Bio, USA) using the XF Mito Stress Test Kit (103015, Agilent, USA). A 96-well XF special cell culture plate was seeded with 2 × 10⁴ cells/well, and the sensor probe plate was hydrated with the seahorse XF calibration solution overnight at 37℃ in a CO₂-free incubator. Then, oligomycin (1.5 µM), 3,3-dioctadecyloxacarbocyanine perchlorate (FCCP) (1.5 µM), and rotenone/antimycin A (0.5 µM) were added successively, and the cells were measured for OCR in the medium (pH: 7.4) containing 2 mM glutamine, 10 mM glucose, and 1 mM pyruvate [48 ].

Mitochondrial oxygen consumption rate (OCR) was measured with the Seahorse XFe96 Extracellular Flux Analyzer (Agilent, Santa Clara, CA, USA), utilizing the Seahorse XF Mito Stress Test kit. After tachypacing, HL-1 cardiomyocytes were trypsinized and 3.5 × 10⁴ cardiomyocytes were plated per well in 0.02% gelatin-coated Seahorse XFe96-well cell culture microplates (Agilent) and incubated at 37 °C and 5% CO₂ for at least 20 h. One hour before the measurement, medium was removed and replaced by DMEM containing 25 mM glucose (Sigma), 1 mM sodium pyruvate (Lonza, Geleen, The Netherlands) and 2 mM l-glutamine (Life Technologies) and cardiomyocytes were incubated in a non-CO₂ incubator at 37 °C. Mitochondrial respiration was measured before (routine) and after 1.5 µM oligomycin (leak respiration), 0.3 µM FCCP (maximal uncoupled respiration) and 2.5 µM antimycin A and 1.25 µM rotenone (residual respiration). Experiments were performed with 8–10 wells per condition and subsequently averaged. OCR values were adjusted for residual respiration and cell count.

The oxygen consumption rate (OCR) and extracellular acidification rate (ECAR) in 4‐day primary CMs were measured with an XF Mito Stress Test Kit (Agilent) using a Seahorse XF^e96 analyzer (Agilent) following the manufacturer's instructions. Prior to the assay, the culture medium was replaced with XF assay medium DMEM supplemented with 10 mM glucose, 2 mM glutamine and 1 mM pyruvate. During the assay, basal OCR and ECAR were measured, followed by sequential injections of 2 μM oligomycin, 3 μM carbonyl cyanide‐p‐trifluoromethoxyphenylhydrazone (FCCP), and 1 μM rotenone/antimycin A through drug injection ports. During the last injection step, cells were simultaneously stained with 2 μM Hoechst 33342 (Thermo Scientific, Rockford, IL, USA) to determine cell number using the Cytation 5 Cell Imaging Multi‐Mode Reader (Biotek, Agilent Technologies). For the ISO treatment experiment, cardiomyocytes isolated from HET(52del) and wild‐type adult hearts were treated with 10 μM ISO for 24 h prior to Seahorse assay. Data were analysed with Seahorse Wave software.