Seahorse xf24

Measurement of the mitochondrial OCR was performed as described previously [27 (link)], using a Seahorse XF-24 extracellular flux analyzer (Seahorse Bioscience, North Billerica, MA, USA). On the day before the experiment, the sensor cartridge was placed into the calibration buffer supplied by Seahorse Bioscience and incubated at 37 °C in a non-CO₂ incubator. Podocytes were cultured on Seahorse XF-24 plates at a density of 20,000 cells per well. The cells were washed and incubated with assay medium (DMEM without bicarbonate) at 37 °C in a non-CO₂ incubator for 1 h. All media and injection reagents were adjusted to pH 7.4 on the day of the assay. Three baseline measurements of the OCR were collected before sequential injection of mitochondrial inhibitors. Three readings were taken after the addition of each mitochondrial inhibitor (before injecting the next inhibitor). The mitochondrial inhibitors used were oligomycin (2 μg/mL), CCCP (10 μM) and rotenone (1 μM). The OCR was automatically calculated and recorded by the Seahorse XF-24 software (Seahorse Bioscience, North Billerica, MA, USA). The percentage change compared with basal rates was calculated as the value of the measurement divided by the average value of the baseline readings.

Metabolic flux analysis was performed as described in ref. 59 (link): Briefly, HepG2 cells were cultured on Seahorse XF-24 (Seahorse BioSciences, Billerica, MA, USA) plates at a density of 7.0 × 10⁴ cells per well. Cells were treated with control vehicle, sesamol (1 mM), rapamycin (100 nM), or sesamol (1 mM) + rapamycin (100 nM). Cells were pre-treated with rapamycin for 30 min and the assays were conducted 24 h post-treatment. On the day of metabolic flux analysis, the media was changed to unbuffered DMEM medium and treated at 37 °C in a non-CO₂ incubator for 1 h. All medium and injection reagents were adjusted to pH 7.4 on the day of assay. Using the Seahorse XF-24 (Seahorse BioSciences) metabolic analyzer, three baseline measurements of OCR and ECAR were sampled prior to sequential injection of mitochondrial inhibitors. Three metabolic determinations were sampled following addition of each mitochondrial inhibitor prior to injection of the subsequent inhibitors. The mitochondrial inhibitors used were oligomycin (4 μM), FCCP (0.2 μM), and rotenone (1 μM). OCR and ECAR were automatically calculated and recorded by the Seahorse X-24 analysis software. After the assays, protein levels were determined by BCA assay for each well to confirm equal cell density per well.

Metabolic parameters were calculated using the Seahorse XF24 as has been described in [59 (link)] and [63 ]. Briefly, BMDM from OPA1^f/f and OPA1^M/M were seeded and polarized in a 24-well Seahorse XF24 cell culture microplate in complete medium and maintained for 24 h at 37 °C, 5% CO₂. To evaluate the Oxygen Consumption Rate (OCR) and extracellular acidification rate (ECAR), one hour previous to the experiment the medium was replaced with Seahorse medium (Dulbecco’s Modified Eagle Medium (DMEM) Sigma-Aldrich) supplemented with 33 mM NaCl, 1 mM sodium pyruvate, 15 mg/l phenol red, and 2 mM glutamine, pH 7.4 and sequentially injecting the following reagents: glucose (25 mM), Oligomycin A (1.5 µM), Carbonyl cyanide-4-(trifluoromethoxy)phenylhydrazone (FCCP) (1.6 µM), Antimycin A (2.5 µM) and Rotenone (1.25 µM). Data were analyzed with Aligent Seahorse Wave software.