Xf96 analyser

The OCR was measured using an XF96 analyser (Seahorse Bioscience, North Billerica, MA, USA). Cells were seeded in 96-well XF96 cell culture plates at a density of 20,000 cells/well. After incubation for 48 h, the cells were treated with C118P (0.025, 0.05, 0.1 μM). The media were then removed, and the wells were washed in XF-modified DMEM (Seahorse Bioscience) at pH 7.4 supplemented with 1 mM glutamine (glycolysis and mitochondrial stress tests), 2.5 mM glucose, 1 mM sodium pyruvate, 0.5 mM carnitine, and 1 mM palmitate in complex with 0.2 mM BSA (mitochondrial stress tests) and incubated for 1 h at 37 °C without CO₂. The OCR was measured in the basal state (1 mM palmitate in complex with 0.2 mM BSA) or after the injection of 5 μM oligomycin, 1 μM 2-[2-[4-(trifluoromethoxy) phenyl] hydrazinylidene]-propanedinitrile (FCCP), and rotenone with antimycin A (both at 0.5 μM). After the Seahorse Bioscience experiments, the proteins were quantified to normalise the results.

Metabolic measurements were done in real-time, non-invasively, using a Seahorse XF96 analyser, which measured the OCR and the ECAR under basal conditions in siDAPK2- or siNS-transfected cells. A cell titration assay was used to determine a suitable cell plating density for both A549 and U2OS cells. For the experiment proper, 0.5 × 10⁶ cells were plated per 10 cm dish and reverse transfected on day 1 as previously described. Forty-eight hours post transfection, cells were re-plated into the XF96 microplates (Seahorse Bioscience; 4 × 10⁴ cells per 100 μl per well) and incubated overnight at 37 °C. The XF calibration solution (Seahorse Bioscience) was added into the XF sensor cartridge (Seahorse Bioscience) and was also incubated at 37 °C overnight but without CO₂. The next day, prior to the assay, complete DMEM was replaced with an XF Assay Medium Modified DMEM (Seahorse Bioscience) (1 g/ml glucose, pH 7.4) and cells were incubated at 37 °C for 1 h without CO₂. Analyses were performed according to the manufacturer's instructions using eight measurements that the instrument recorded for OCR (nmoles/min) and ECAR (mpH/min) pertaining to each well. Results were analysed using the provided XFe Wave software (Seahorse Bioscience).

Seahorse XF96 Cell Mito stress test was performed as described in the manufacturer protocol. In brief, B cells of 30 APs and 3 HDs were isolated from the cryopreserved PBMC vials. B cells of 30 APs were pooled together to obtain sufficient signal. A total of .5 × 10⁶ purified B cells per well were resuspended in a 180‐μl Seahorse medium (unless otherwise specified) and plated in a 96‐well Cell‐Tak (Corning)‐coated Seahorse plate. Cells were maintained in 37°C in a non‐CO₂ incubator for at least 1 h before the assay. Cells were treated sequentially with 1.5‐μM oligomycin (an inhibitor of ATP synthase), 10‐μM carbonyl cyanide‐p‐trifluoromethoxyphenylhydrazone (FCCP, an uncoupler of mitochondrial oxidative phosphorylation) and 5‐μM rotenone plus 5‐μM antimycin A (inhibitors of mitochondrial electron transport chain). Oxygen consumption rate (OCR) was measured using a Seahorse XF96 analyser.

Oxygen consumption rates (OCR) and extracellular acidification rates (ECAR) were determined using a Seahorse XF96 Analyser (Seahorse Bioscience). Respiratory profiles were generated by serial treatment with optimised concentrations of oligomycin (1 μg/mL), p-[trifluoromethoxy]-phenyl-hydrazone (FCCP, 500 nM) and rotenone (500 nM). Glycolytic profiles were generated by serial treatment of glucose-restricted cells with optimised concentrations of glucose (12.5 mM), oligomycin A (1 μM), and 2-DG (50 mM). Cell number normalisation was carried out post-respirometry using sulforhodamine B (SRB) staining of TCA-fixed cells in the assay plate.

OCR was determined using a Seahorse XF96 Analyser (Seahorse Bioscience). Respiratory profiles were generated by serial treatment with optimised concentrations of oligomycin (1 µg/mL), p-[trifluoromethoxy]-phenyl-hydrazone (FCCP, 500 nM), and rotenone (500 nM). Cell number normalisation was carried out post-respirometry using sulforhodamine B (SRB) staining of TCA fixed cells in the assay plate.