Xf dmem medium

RAW 264.7 (R0 and RMA) cells were seeded in Seahorse XF24 cell culture microplates (75 × 10³ cells/well) in growth medium (DMEM with high glucose and 10% foetal bovine serum). For the twelve-hour treatment, cells were treated with LPS (100 ng/mL) or the vehicle 36 h after seeding. The growth medium was replaced with the assay medium (Seahorse XF DMEM medium supplemented with glucose (10 mM), glutamine (2 mM), and pyruvate (1 mM)) and LPS (100 ng/mL)) or the vehicle 11 h later. Cells were then incubated for an additional 60 min at 37 °C without CO₂ before being subjected to the mito stress test. For the four-hour treatment, cells were treated with LPS (100 ng/mL) or the vehicle 48 h after seeding. The growth medium was replaced with an assay medium containing LPS (100 ng/mL) or the vehicle 3 h later. Cells were incubated for an additional 60 min at 37 °C without CO₂ prior to the mito stress test. The mito stress test was performed using the Seahorse XFe24 Analyser (Agilent) with oligomycin (1.5 μM), carbonyl cyanide-p-trifluoromethoxyphenylhydrazone (FCCP) (1.5 μM), and rotenone (0.5 μM) + antimycin A (0.5 μM). After analysis in Seahorse, cells were lysed with 0.1% SDS in water, and the protein content was measured using the BCA protein assay. All OCR and ECAR measurements were normalised to the protein content prior to further analysis.

0.5x10⁵ BMDMs were seeded in a 96-well Seahorse plate. Following treatments cells were washed and incubated with Seahorse XF DMEM Medium pH 7.4 media supplemented with 10 mM glucose, 1 mM sodium pyruvate, and 2 mM glutamine. Seahorse extracellular flux analysis measured oxygen consumption rate (OCR) (pmol/min) and extracellular acidification rate (ECAR) (mpH/min). ECAR was converted into proton efflux rate (PER) (pmolH+/min), a more accurate measure of extracellular acidification, by Seahorse software. Addition of mitochondrial stress test compounds (5 µM oligomycin, 10 µM FCCP, 5 µM rotenone/antimycin) and glycolytic rate assay compounds (5 µM rotenone/antimycin a, 5 mM 2-DG) were made to derive different measures of metabolism.

BMDMs were seeded in Seahorse XF24 cell culture microplates (2.2 × 10⁵ cells/well) in growth medium (DMEM with high glucose and 20% FBS). For the 12-h treatment: 36 h after seeding, cells were treated with LPS (100 ng/mL) or vehicle; 11 h later, growth medium was replaced with assay medium (Seahorse XF DMEM medium supplemented with glucose (10 mM), glutamine (2 mM) and pyruvate (1 mM)) with LPS (100 ng/mL) or vehicle. Cells were incubated for an additional 60 min at 37 °C without CO₂ before a mito stress test. For the 4-h treatment: 48 h after seeding, cells were treated with LPS (100 ng/mL) or vehicle; 3 h later, growth medium was replaced with assay medium with LPS (100 ng/mL) or vehicle. Cells were incubated for an additional 60 min at 37 °C without CO₂ before a mito stress test. The mito stress test was performed in Seahorse XFe24 Analyzer (Agilent) with oligomycin (1.5 μM), FCCP (1.5 μM) and rotenone (0.5 μM) + antimycin A (0.5 μM). After analysis in Seahorse, cells were lysed with 0.1% SDS in water and analyzed for protein content with BCA protein assay. All OCR and ECAR measurements were normalized to protein content before any further analysis.

On the day of the assay, cells were washed twice with seahorse media (Seahorse XF DMEM Medium (pH 7.4) supplemented with 2 mM L-glutamine, 1 mM sodium pyruvate, and 25 mM glucose), before 175 μL fresh seahorse media was added and cells were equilibrated in a non-CO₂ incubator at 37 °C for one hour before beginning the assay. The Seahorse mitochondrial stress test assay was used, consisting of consecutive additions of oligomycin (1 μM), FCCP (0.25 μM), and rotenone/antimycin A (1 μM) (concentrations chosen following optimization assays). Rates of oxygen consumption and extracellular acidification were recorded at regular intervals throughout the assay. Parameter calculations were performed using the Seahorse XF Cell Mito Stress Test Report Generator.