Seahorse xfe96 extracellular flux analyzer

The real-time measurement of the oxygen consumption rate (OCR) was performed by an XFe96 Seahorse Extracellular Flux Analyzer (Agilent, Santa Clara, CA, USA) by using the MitoStress Kit (Agilent) according to the manufacturer’s procedures. A total of 80,000 cells/well were seeded, and the OCR was measured in XF media (non-buffered RPMI medium containing 10 mM glucose, 2 mM L-glutamine, and 1 mM sodium pyruvate) under basal conditions and in response to 2 μM oligomycin, 1.5 μM of carbonylcyanide-4-(trifluoromethoxy)-phenylhydrazone (FCCP), and 1 μM of Antimycin and Rotenone (all from Sigma Aldrich). Indices of the mitochondrial respiratory function were calculated from the OCR profile: basal OCR (before the addition of oligomycin), ATP-linked OCR (calculated as the difference between the basal OCR rate and oligomycin-induced OCR rate), maximal OCR (calculated as the difference of the FCCP rate and Antimycin plus Rotenone rate), spare respiration capacity (SRC, the difference between the basal ATP production and its maximal activity), and non-mitochondrial oxygen consumption. Each experiment was repeated three times, each in triplicate.

Metabolic assays were performed using the XF^e96 Seahorse extracellular flux analyzer (Agilent Technologies). Anti-CD3:anti-CD28 stimulated and unstimulated CD4⁺ T cells were plated at 4.5 × 10⁵ live cells/well in plates coated with Cell Tak (Corning #354240). Final well drug concentrations for the Mito Stress Test (Agilent #103015-100) were as follows: Oligomycin 2 μΜ, FCCP (Carbonyl cyanide-4-phenylhydrazone) 2 μM, and Rotenone/Antimycin A 0.5 μΜ. Culture media for the Mito Stress Test contained RPMI and 2 mM glutamine without a buffer solution (Sigma #R1383), 10 mM glucose (Sigma #S8636) and 1 mM pyruvate (Sigma #G8769). Equations for parameters and a graphical representation of calculations from the report generator used for Mito Stress Test can be found in Supplementary Fig. S2. Final well concentrations for the Glycolysis Stress Test (Agilent #103020-100) were as follows: Glucose 10 mM, Oligomycin 2 μM, 2-Deoxyglucose 50 mM. Culture media for the Glycolysis Stress Test was RPMI containing 2 mM glutamine without a buffer solution (Sigma #R1383) and no glucose or pyruvate. Equations for parameters and a graphical representation of calculations from the report generator used for Glycolysis Stress Test can be found in Supplementary Fig. S3.

Glucose and lactate levels in the medium were determined through routine diagnostic procedures by the department of Clinical Chemistry of the AMC Amsterdam. For the CS activity assay BMDM cells were collected and lysed in 0.5% (w/v) Triton X-100, and activity was determined as described previously [34 ]. For ATP measurements, BMDM were cultured in 24-well plates. One hour prior, the assay control cells were incubated with 5 mM 2-Deoxy-D-glucose and 2.5 µm AntimycinA (Sigma-Aldrich). ATP levels were determined using a kit (ThermoFisherScientific) according to the manufacturer’s protocol. Cs activity, glucose, lactate, and ATP values were adjusted for total protein concentration as determined using BCA (Sigma-Aldrich). Cellular OCR and ECAR were determined via real-time extracellular flux analysis using the XFe96 Seahorse Extracellular Flux Analyzer (Agilent, Santa Clara, CA, USA) in BMDM plated and polarized in XF 96-well assay plates 48 h before the assay. OCR and ECAR were determined with the extracellular flux assay according the protocol of Van den Bossche et al. [30 ]. ECAR and OCR values were adjusted for cell input using the CyQUANT^® Cell Proliferation Assay Kit (ThermoFisherScientific) according to the supplier’s protocol.

Oxygen consumption rates (OCRs) were determined by Seahorse Extracellular Flux Analyzer XFe96 (Seahorse Bioscience, Billerica, MA, USA). Human islets, in groups of 10, were preincubated for 3 h at 20 mM glucose before the start of analysis. After 20 min of recording at basal conditions, GLX7013114 (0.5 and 2 μM) was injected into some of the wells. After another 20 min, Oligomycin, FCCP, and Antimycin + rotenone were consecutively injected at 20–30 min intervals.

Metabolic analysis of T cells was carried out using a Seahorse Extracellular Flux Analyzer XF^e96 (Seahorse Bioscience). Mitochondrial respiration was measured by oxygen consumption using a XF Cell Mito Stress Test kit (Seahorse Bioscience). Pan T cells were isolated from spleens of 7-week-old female C57BL/6J mice using a Pan T Cell Isolation kit II (Miltenyi Biotec). T cells were incubated with plate-bound anti-CD3 (1 µg ml⁻¹) and soluble anti-CD28 (2 µg ml⁻¹), and stimulated with IL-2 (100 ng ml⁻¹) and IFNγ (1 ng ml⁻¹) with or without AC484 (20 µM) for 3 days. T cells were seeded at a density of 495,000 per well in a poly-l-lysine-coated Seahorse XF96 Cell Culture Microplate in RPMI 1640 base medium (US Biological, R9011, pH 7.4) supplemented with 10 mM glucose, 2 mM glutamine and 1 mM sodium-pyruvate (all Sigma-Aldrich). Assays were performed according to the manufacturer’s instructions using 1.5 µM oligomycin to inhibit ATP synthase, 1 µM FCCP to uncouple oxygen consumption from ATP production and 0.5 µM rotenone and antimycin A to stop the electron transport chain. Data were analysed using Wave Software (Seahorse Bioscience). Using Prism, the data were statistically assessed using a multiple unpaired t-test or a two-way analysis of variance.