Seahorse xf rpmi medium

AMs were isolated by BAL six days after in vivo training. Biological replicates were pooled by experimental group and seeded as technical replicates in XF-96 cell culture plates (Agilent) at a density of 8 × 10⁴ cells/well in 80 µL RPMI medium (3% FCS, 1% PS). To remove non-adherent cells, the plate was incubated for 2 h at 37 °C and cells were washed twice either with PBS (followed by subsequent ex vivo challenge, performed as described) or XF assay medium (Seahorse XF RPMI medium, pH 7.4, 10 mM Glucose, 1 mM Pyruvate, 2mM L-Glutamine [all from Agilent], 3% FCS). Prior to analysis, cells were incubated under non-CO₂ conditions in XF assay medium for 1 h. Oxygen consumption rate (OCR) and extracellular acidification rate (ECAR) of AMs were analyzed using a Seahorse XF-96 Extracellular Flux Analyzer (Agilent). Where indicated, 1 μM oligomycin, 1.5 μM carbonyl cyanide p-trifluoromethoxyphenylhydrazone (FCCP) or 100 nM rotenone plus 1 μM antimycin A (R/A; all from Sigma) were injected to assess mitochondrial function. Means of R/A values (non-mitochondrial respiration) were subtracted from OCR raw data for quantification of mitochondrial parameters. ECAR data represent raw values.

Metabolic phenotypes of PBMCs from 7 WT pigs (5 females, 2 males) and 6 GHR-KO pigs (4 females, 2 males) were determined in three independent experiments using a Seahorse XFe Analyzer (Agilent Technologies, Waldbronn, Germany). Oxygen consumption rate (OCR) was measured, indicating mitochondrial respiration and extracellular acidification rate (ECAR) reflected glycolysis [42 (link)]. Sterile XF assay buffer (Seahorse XF RPMI medium supplemented with 10 mM glucose, 2 mM L-glutamine, and 1 mM pyruvate, pH 7.4; Agilent Technologies) was used for the experiments according to the manufacturer’s instructions. Before starting the assay, sensor cartridges (Agilent Technologies) were prepared to which oligomycin, carbonyl cyanide-4-(trifluoromethoxy)-phenylhydrazone (FCCP), and rotenone were added along with antimycin A. The cartridges were then used for the experiments. A total of 1 × 10⁶ PBMCs were seeded in 24-well XF24 cell culture microplates (Agilent Technologies), while four wells were kept free from cells for background correction. Baseline OCR and ECAR were measured before adding oligomycin, FCCP, and rotenone together with antimycin A. OCR was reported in units of pmol/minute and ECAR in mpH/minute.

Real‐time measurements of oxygen consumption rate (OCR) and extracellular acidification rate (ECAR) were performed using a Seahorse XF96 Extracellular Flux Analyzer (Agilent Technologies). Donor‐derived human osteoclasts were cultured for 7 days and sorted as mononuclear and multinuclear cells (see description above). Cells were then washed and 5 × 10⁵ osteoclasts were seeded onto a XF96 plate containing Seahorse XF RPMI medium (Agilent Technologies). The cells were left for 1 h at 37°C after which the different metabolic drugs were injected (oligomycin 1 μM, FCCP 2 μM, rotenone/antimycin 1 μM, 2‐DG 50 mM) during real‐time measurements of OCR and ECAR, using the Seahorse XF Cell Mito Stress Test Kit (Agilent Technologies). Basal respiration was calculated as [the last measurement before addition of oligomycin—non‐mitochondrial respiration (minimum rate measurement after Rot/AntA)]. Maximal respiration is shown as [the maximum rate measurement after addition of FCCP—non‐mitochondrial respiration]. Estimated ATP production designates [the last measurement before addition of oligomycin—minimum rate after oligomycin]. Glycolysis refers to ECAR values before the addition of oligomycin.

Metabolic phenotypes of PBMC from 15 wild-types and 10 INS^C94Y tg pigs were determined in five independent experiments, using a Seahorse XFe Analyzer (Agilent Technologies, Waldbronn, Germany) measuring oxygen consumption rate (OCR), which is attributed to mitochondrial respiration and extracellular acidification rate (ECAR), which can be related to glycolysis (20 (link)). Duplicates (technical replicates) were generated for each animal. Mean values of technical replicates were then used for further statistical analysis. In accordance with the manufacturer’s instructions, sterile XF assay buffer (Seahorse XF RPMI Medium supplemented with 10 mM glucose, 2 mM L-glutamine, and 1 mM pyruvate, pH 7.4; Agilent Technologies, Waldbronn, Germany) was used for experiments. Prior to the start of the assay, sensor cartridges (Agilent Technologies, Waldbronn, Germany) were prepared adding oligomycin, FCCP and rotenone & antimycin A. A total of 1 x 10⁶ PBMC was seeded in 24-well XF24 cell culture microplates (Agilent Technologies, Waldbronn, Germany), while four wells were kept free from cells as background correction. Baseline OCR and ECAR were measured before adding oligomycin, FCCP and rotenone & antimycin A. OCR was reported in units of pmol/minute and ECAR in mpH/minute.

CD4+ T cells were cultured as indicated for 72 h, after which they were washed and resuspended in Seahorse XF RPMI medium (Agilent Technologies; pH 7.4, supplemented with 1 mM HEPES, 25 mM glucose, 1 mM pyruvate, 2 mM glutamine) at 5x10⁶ cells/mL. 2x10⁵ cells were added to each well of a 96-well assay plate, pre-coated with poly-D-lysine. Each sample was added to four wells to provide technical replicates. Mitochondrial respiration was measured by oxygen consumption rate (OCR) and glycolytic rate by extracellular acidification rate (ECAR), using a Seahorse XFe96 Extracellular Flux Analyzer (Agilent Technologies) with analysis using the Seahorse XF Cell Energy Phenotype report generator. Metabolic activity was measured in the presence of glucose at the indicated time points, under resting conditions (first three data points) and under stress conditions following addition of 1 μM oligomycin and 1 μM carbonyl cyanide 4-(trifluoromethoxy)phenylhydrazone (FCCP; Agilent Technologies).

T cells were resuspended in serum-free Seahorse XF RPMI medium (Agilent, Lexington, MA, USA) and then plated into Seahorse XF cell culture microplates (1.5 × 10⁵ cells per well) coated with poly-D-lysine (Sigma-Aldrich, Oakville, ON, Canada) for T-cell attachment. A mitochondrial stress test was performed on a Seahorse XF analyzer (Agilent, Lexington, MA, USA) to measure OCR (pmol min⁻¹) under basal conditions and upon sequential injection of oligomycin (1.5 μM), FCCP (2.5 μM), and rotenone/antimycin A (0.5 μM) (Agilent, Lexington, MA, USA) [18 (link)]. The following conditions were used in experiments with the Seahorse system: 3 min mixture; 0 min wait; and 3 min measurement.