Seahorse xf dmem medium ph 7

HEK293T cells were transfected with the left and right halves of DdCBE fused to GFP and mCherry through a self-cleaving T2A sequence. Three days after transfection, cells that express both halves of the DdCBE were enriched by fluorescence-activated cell sorting (FACS) using BD FACSAria III. The FACS gating strategy is shown in Supplementary Note 3. The sorted cells were recovered three generations (passaged every other day) before measuring the oxygen consumption rate. Oxygen consumption rates of HEK293T cells were measured using a Seahorse XF24 Extracellular Flux analyzer (Agilent) following the manufacturer’s instruction. In brief, 45,000 cells were seeded on the Seahorse plate coated with 0.01% (w/v) poly-d-lysine for 16 h. Analysis was performed in Seahorse XF DMEM Medium pH 7.4 (Agilent, 103575-100) supplemented with 10 mM glucose (Agilent, 103577-100), 2 mM l-glutamine (Agilent, 103579-100), and 1 mM sodium pyruvate (Sigma, S8636). Mito stress protocol was applied with the use of 1 μM oligomycin, 0.5 μM FCCP, and 1 μM rotenone + antimycin A.

The mitochondrial activity of differentiated WT and NTRK2^–/– ReNcell VM was measured using Seahorse XF Cell Mito Stress Test (Agilent). For this, 30 000 cells were plated into Seahorse XF Cell Culture Plates and differentiated for 5 days. The XFe96 cartridge was hydrated at 37°C overnight. On the following day, water was replaced by pre-heated Seahorse XF Calibrant for 90 min. After removal of the differentiated medium, 180 μl Agilent Seahorse XF DMEM Medium pH 7.4 (# 103575–100) was added to each well and kept for 1 h at 37°C. Oxygen consumption was measured. After 17 min, oligomycin (3 μM final) was added to block ATP-synthase, leading to decreased to mitochondrial oxygen consumption. After another 17 min, the uncoupling compound carbonyl cyanide-4 (trifluoromethoxy) phenylhydrazone (FCCP) (1 μM final) was added to the cells leading to maximal oxygen consumption. Finally, mitochondria activity was poisoned by addition of a mix of rotenone and antimycin A (0.5 μM final) to measure non-mitochondrial oxygen consumption.

A Seahorse plate was coated with 0.01% (w/v) poly-l-lysine (Sigma), and 1.5 × 10⁴ cells per well were seeded 16 h before analysis with the Seahorse Xfe96 Analyzer (Agilent). Analysis was performed in the Seahorse XF DMEM Medium pH 7.4 (Agilent), supplemented with 10 mM glucose (Agilent), 2 mM l-glutamine (Gibco) and 1 mM sodium pyruvate (Gibco). The Mito stress protocol was applied using 1.5 μM oligomycin, 1 μM FCCP and 1 μM piericidin + 1 μM antimycin. After the run, media was removed from the wells, and cells were stained with Hoechst 33342 (Thermo Fisher Scientific; final 1:5,000 dilution in PBS) for 15 min at room temperature. Next, the staining solution was removed, and cells were imaged in PBS using the BioTek Cytation 5 Cell Imaging Multimode Reader (Agilent). The oxygen consumption rate values were normalized to the number of cells per well, calculated as imaged nuclei in each well.

1 d before the experiment, 40,000 cells were seeded per well into 96-well cell culture microplates (Seahorse XF96, V3-PS, TC-treated; Agilent Technologies). The extracellular flux assay kit (Seahorse XFe96; Agilent Technologies) cartridge was hydrated with 200 μl XF Calibrant and sealed in a bag with a wet towel and left in a non-CO₂ incubator overnight. The next day, cell confluency reached 90–100%. The cell plate was placed in the non-CO₂ incubator at 37°C for 1 h before the experiment. The assay medium contained the Seahorse XF DMEM medium pH 7.4 (Agilent Technologies) supplemented with 2.5 mM glucose, 1 mM pyruvate, and 2 mM L-glutamine. Injection solution A contained a final concentration of 1 μm oligomycin, injection B 1 μM FCCP, injection C 1 μM rotenone, 1 μM antimycin A and 50 μg/ml Hoechst solution. The data were normalized to the cell number calculated by Hoechst staining at the end of the experiment.

Glycolysis was assessed by measuring oxygen consumption rate and extracellular acidification rate (ECAR) using a Seahorse XFe96 analyzer (Agilent Technologies) at the Biomarkers Platform at Pasteur institute (Paris), to determine the PER and PER associated to glycolysis only (glycoPER). Briefly, MeWo and A375M cell lines were transfected as describe above 48 h before the assay and were seeded on Seahorse XF96 cell culture microplate (Agilent Technologies) at a density of 10,000 cells/well 24 h before the assay. The day of the assay, the medium was replaced by Seahorse XF DMEM medium, pH 7.4 (Agilent Technologies), supplemented with 1 mM pyruvate (Agilent Technologies), 2 mM glutamine (Agilent Technologies), and 10 mM glucose (Agilent Technologies) and incubated 1 h in a non-CO₂ incubator. Just before the assay, cell medium was changed for fresh warm assay medium. A final well concentration of 0.5 μM of rotenone and antimycin A mixture and 50 mM of 2-deoxy-D-glucose (2-DG) were sequentially injected to calculate PER and glycoPER. Data were analyzed using the Wave software 2.6.3 (Agilent Technologies) and normalized to cell confluency (in %) obtained by phase-contrast imaging and analyzed with Incucyte SX5 (Sartorius). Curves were plotted using RStudio; superplots and statistical analysis were generated using SuperPlotsofData (Goedhart, 2021 (link)).

The XF96 Extracellular Flux Analyzer (Agilent, Santa Clara, CA) was used to determine the glycolytic profile of cells. Oxygen consumption rate (OCR), extracellular acidification rate (ECAR) and proton efflux rate (PER) were determined using the Glycolytic Rate Assay Kit (Agilent), according to the protocols provided by the manufacturer. Briefly, macrophages were seeded at a density of 1 x 10⁵ cells per well in the manufacture’s 96-well plate and allowed to attach overnight. Cells were then infected or not with B. abortus at a MOI of 100:1 for 24 hrs in normal cell culture medium. Prior to the assay, cells were washed tree times and media was changed to Seahorse XF DMEM medium pH 7.4 (Agilent), supplemented to contain 25 mM glucose, 4 mM L-glutamine and 2 mM pyruvate. The plate was allowed to equilibrate for 1 hr in a CO₂-free incubator at 37 °C before loading into the Seahorse analyzer. Seahorse XF96 cartridges were hydrated according to the manufacturer’s instructions. Three measurements of OCR and ECAR were performed before injection of mitochondrial inhibitors (rotenone and antimycin A) and then three additional measurements were achieved before injection of the inhibitor 2-DG. Experiments were performed with 4 replicates of each condition. The bioenergetic parameters mitoPER and glycoPER were calculated as described elsewhere [34 (link)].