Seahorse xf cell culture plates

The assessment of oxygen consumption rate (OCR) in U2-OS cell lines was undertaken as described [41 (link)]. Briefly, 5 × 10⁴ cells/well of each U2-OS cell line were seeded in 24-well Seahorse XF Cell Culture Plates in 500 μL of complete medium and left for 24 h at 37 °C. The Seahorse XFe24 Sensor Cartridges were filled with Seahorse XF Calibrant Solution pH 7.4 and left overnight to hydrate in a 37 °C incubator without CO₂. Just prior to the experiment all the growth medium was removed and the cells were washed with fresh Seahorse Assay medium (Seahorse XF DMEM Medium, pH 7.4 supplemented with 10 mM glucose, 1 mM pyruvate and 2 mM l-glutamine) pre-warmed at 37 °C. A final volume of 500 μL Seahorse Assay medium was left in the wells for the experiment. To equilibrate temperature and pH the plate was incubated in a 37 °C incubator lacking CO₂ for 1 h prior to the assay. To generate OCR, oligomycin (1 μM), FCCP (1 μM) and antimycin A (0.1 μM) were injected in the wells by the Seahorse XFe24 Analyzer and 3 measurements of OCR were taken in each cycle of the analysis. At the end of the assay the medium was removed and the cells were washed with cold PBS and lysed with RIPA buffer for protein quantification. Protein was quantified using a BCA assay. The final values were calculated and expressed with normalization of protein content using Agilent software Wave.

HLC mitochondrial function was assessed by direct measurement of the oxygen consumption rate (OCR) using extracellular flux analysis (XF24, Seahorse Biosciences, North Billerica, MA, USA). HLCs were inoculated in pre-coated Seahorse XF cell culture plates as described in section Cell culture’ methods section. The Mitochondrial Stress Test assay was performed in XF Base medium (Agilent Technologies, Santa Clara, CA, USA) with 1 mM of pyruvate, 2 mM of L-glutamine (ThermoFisher Scientific, Waltham, MA, USA) and 10 mM of glucose. Baseline OCR were measured every 7 minutes. Following baseline measurements, oligomycin (1.5 µM), Carbonyl cyanide-4-(trifluoromethoxy)phenylhydrazone (FCCP) (1.25 µM), and rotenone (2 µM) and antimycin A (2 µM) were sequentially injected to measure OCR. From the obtained OCR profile, basal respiration, ATP production, maximal respiration and spare respiratory capacity could be calculated. Protein extraction from the HLCs was performed with RIPA lysis buffer supplemented with protease inhibitors 1x (Roche, Basel, Switzerland). Protein concentration (µg/µL) was determined using the bicinchoninic acid (BCA) protein assay kit (Pierce) according to manufacturer instructions. Mitochondrial function was normalized to the protein concentration (µg/µL) of each well.

Cells were seeded in six-well plates (200,000 cells/well) and transfected appropriately, as described in figure legends. The day before the assay, cells were detached by trypsinisation, counted, and seeded at 10,000 cells/well in 96-well Seahorse XF cell culture plates (Agilent). The sensor cartridges were hydrated overnight with tissue culture grade H₂O in a non-CO₂ incubator at 37°C as per the manufacturer's instructions. On the day of the assay, H₂O in the sensor plate was replaced with Seahorse XF Calibrant (Agilent) and cells were washed with HBSS and incubated in Seahorse medium [Seahorse XF assay medium (Agilent), 1 mM pyruvate (Agilent), 2 mM glutamine (Agilent) and 10 mM D-(+)-galactose]. Both sensor and cell plates were incubated in a non-CO₂ incubator at 37°C for 1 h before the assay. The sensor plate was loaded with oligomycin (15 µM for 1.5 µM final concentration in wells; injector A), CCCP (5 µM for 0.5 µM final; injector B), and antimycin A and rotenone (5 µM/5 µM for 0.5 µM/0.5 µM final; injector C). The sensor plate was calibrated before loading cells and running a mitochondrial stress test using a Seahorse XFe96 (Agilent). Following assays, cells were washed and fixed in 1% (v/v) acetic acid in methanol at −20°C overnight for sulforhodamine B (SRB) assays, which were used for normalising data to protein content.