Xf base medium minimal dmem

Mitochondrial respiration was assayed in fibroblasts treated or not treated with 2DG on 24-well XF24e plates, using an XF24e Extracellular Flux Analyzer (Agilent Technologies). Briefly, 5 × 10⁴ cells were seeded approximately 16–24 h before the assay in prewarmed growth medium (DMEM, Gibco) and incubated at 37 °C. Subsequently, the medium was removed and replaced with assay medium (XFBase medium minimal DMEM (Agilent) complemented with 2 mM glucose, 2 mM glutamax and 1 mM pyruvate) and cells incubated for 30 min in a 37 °C non-CO₂ incubator. After taking an oxygen consumption rate (OCR) baseline measurement, 1 µM oligomycin, 0.75 µM carbonylcyanide-4-trifluorometho-xyphenylhydrazone (FCCP) and 1 µM rotenone were added sequentially. For the extracellular acidification rate (ECAR) values, the average of the first three measurements of the basal level prior the oligomycin injection was considered.

The oxygen consumption rate and extracellular acidification rate were measured by using a flux analyzer (Seahorse Bioscience XF24 Extracellular Flux Analyzer, Agilent, Santa Clara, CA, USA) and XF Mito Stress Kit (Agilent). 3T3L1 cells were seed at 3.5 × 10³ cells per well in 0.1% gelatin-coated Seahorse 24-well plates and differentiated into adipocytes as described above. After differentiation, B. wexlerae culture supernatant or uncultured fresh medium were added to the 3T3L1 adipocyte cultures to a final concentration of 10%; cultures were then incubated at 37 °C for 1 h. After incubation, the culture medium was changed to XF Base Medium Minimal DMEM (Agilent) supplemented with 10 mM glucose (Nacalai Tesque), 1 mM pyruvate (Nacalai Tesque), and 2 mM l-glutamine (Nacalai Tesque) for measurement. Compounds injected during the assay and their final concentrations were 1.5 µM oligomycin (inhibitor of ATP synthase), 1 µM FCCP (proton uncoupling agent), and 0.5 µM rotenone + 0.5 µM antimycin A (inhibitors of the mitochondrial respiration complex). XFe Wave software (Agilent) was used to analyze the results.

Mitochondrial respiration was assayed by measuring the oxygen consumption rate (OCR) in control or SERBP1 overexpressing U343 glioblastoma cells using a Seahorse XFe96 Analyzer (Agilent Technologies). Cells were plated at a concentration of 15,000–20,000 cells/well into XF96 Seahorse plates 2 days before the experiment. Media was changed the next day to low serum media (containing 2%FBS) and the experiment was run the next day using XF Base Medium Minimal DMEM (Agilent Technologies) supplemented with 1 mM sodium pyruvate, 5.5 mM glucose, and 2 mM glutamine. Injections were prepared following the manufacturer’s protocol for the Mito Stress Kit (Agilent Technologies). OCR was normalized to confluence using an IncuCyte® ZOOM phase-only processing module (Essen Bioscience).

Twenty five thousand monocytes per well were seeded in a Seahorse XFp cell culture plate and primed as previously described. The oxygen consumption rate (OCR) was evaluated under basal conditions and in response to 2 μM oligomycin, 1.5 μM FCCP, 100 nM rotenone, plus 1 μM antimycin A (all from Sigma-Aldrich). The cells were cultured in XF-medium (XF Base Medium Minimal DMEM; Agilent Technologies) containing 10 mM glucose, 2 mM l-glutamine, and 1 mM sodium pyruvate (all from Sigma-Aldrich). OCR was determined using a Seahorse XFp Analyzer (Agilent), and assays were analyzed with Wave Desktop software (Agilent), as described before (19 (link)).

Data were analyzed as change in OCR and ECAR as a function of SGLT2 inhibitor concentration. Effects on cellular respiration and medium acidification was measured with Agilent/Seahorse Extracellular Flux analyzer XF96 at 37 °C. Basal OCR and ECAR were recorded prior to injection of the respective SGLT2 inhibitor to yield final concentrations of 0.2, 1, 5, 25, and 100 µM (n = 4), followed by five repeated measurements of OCR and ECAR (during approximately 35 min). Rotenone (2 µM) was used as reference for the inhibition of the mitochondrial ETC complex I. DMSO was utilized as blank sample. In total, 15,000 and 25,000 HUVEC cells were seeded in 150-microliter cell culture medium per XF96 plate well 18–24 h prior to experiment. XF96 cartridge was equilibrated with calibration buffer overnight according to manufacturer´s instructions. Cell culture medium was changed to XF Base Medium Minimal DMEM (Agilent, Santa Clara, CA, USA) complemented with 5 mM glucose, 1 mM pyruvate, 1 mM glutamine, and 15 mM HEPES 30–40 min prior to first reading. After the last read-out, cells were fixed in 4% formaldehyde, and subsequently washed with 4% formaldehyde prior to adding Hoechst nuclear stain for cell counting with Image Xpress system from Molecular Devices. OCR and ECAR were normalized based on the cell numbers per well.

For 72 h, ESdMs were incubated with LPS ± NaCl (40mM) to stimulate cells. Oxygen consumption rate (OCR) was determined under basal conditions and in response to 2 μM oligomycin, 1.5 μM FCCP, 100 nM rotenone plus 1 μM antimycin A (all Sigma-Aldrich). During OCR measurement activated ESdMs were cultured in XF-medium (XF Base Medium Minimal DMEM (Seahorse)) containing 10 mM glucose, 2 mM L-glutamin, and 1 mM sodium pyruvat (all from Sigma-Adlrich). OCR were evaluated using the Seahorse XFp Extracellular Flux Analyzer (Agilent Technologies) and analysed with Wave Software (Seahorse Bioscience).