Real-time measurement of oxygen consumption rate (OCR) and extracellular acidification rate (ECAR) were monitored using Vero cells infected with RVPs, with the XFe extracellular flux analyzer (Agilent Seahorse Technologies, Santa Clara, CA, USA). Briefly, 10,000 Vero cells/well were plated into XF96 microplates for ECAR measurement, and 15,000 cells/well for OCR measurement. Prior to the analysis, media was changed to XF DMEM (Agilent Seahorse Technologies, Santa Clara, CA, USA) supplemented with 10 mM glucose (Agilent Seahorse Bioscience), 1 mM sodium pyruvate and 2 mM L-glutamine for OCR (readout of mitochondrial respiration) and to XF DMEM (Agilent Seahorse Technologies, Santa Clara, CA, USA) supplemented with 2 mM of glutamine for ECAR monitorization as an indirect readout of acidification by the production of lactate (aerobic glycolysis). For ECAR measurement the following sequential injections were performed: glucose (10 mM), oligomycin (2 µM), and 2-DG (75 mM). For OCR, sequential injection of oligomycin (2 µM), trifluoromethoxy carbonylcyanide phenylhydrazone (FCCP; 1.2 µM), and rotenone/antimycin A (0.5 µM) as previously described [21 (link), 22 (link)].
Xf dmem
Manufactured by Agilent Technologies
Sourced in United States
XF DMEM is a cell culture medium designed for use in Seahorse XF Analyzers. It is formulated to optimize the measurement of cellular metabolic parameters, such as oxygen consumption rate and extracellular acidification rate, in a variety of cell types.
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Lab products found in correlation
Fetal bovine serum (fbs), by Thermo Fisher Scientific (4 mentions)
Bca protein assay kit, by Thermo Fisher Scientific (4 mentions)
Penicillin streptomycin, by Thermo Fisher Scientific (4 mentions)
Seahorse xfe96 analyzer, by Agilent Technologies (3 mentions)
High capacity cdna reverse transcription kit, by Thermo Fisher Scientific (3 mentions)
Xfe extracellular flux analyzer, by Agilent Technologies (2 mentions)
Xf ps 96 well plates, by Agilent Technologies (2 mentions)
Rneasy mini kit, by Qiagen (2 mentions)
Power sybr green pcr master mix, by Thermo Fisher Scientific (2 mentions)
Phosphate buffered saline (pbs), by Thermo Fisher Scientific (2 mentions)
Xfe24 extracellular flux analyzer, by Agilent Technologies (1 mentions)
Seahorse xf cell mito stress test kit, by Agilent Technologies (1 mentions)
Xfe96 analyzer, by Agilent Technologies (1 mentions)
Cell tak cell and tissue adhesive, by Corning (1 mentions)
Seahorse xf96 analyzer, by Agilent Technologies (1 mentions)
Xfp extracellular flux analyzer, by Agilent Technologies (1 mentions)
Cell energy phenotype test kit, by Agilent Technologies (1 mentions)
Agilent seahorse xfe24 analyzer, by Agilent Technologies (1 mentions)
Seahorse xf glutamine solution, by Agilent Technologies (1 mentions)
Seahorse xf pyruvate solution, by Agilent Technologies (1 mentions)
17 protocols using xf dmem
1
Aerobic Respiration and Glycolysis Measurement
2
Aerobic Respiration and Glycolysis Measurement
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Real-time measurement of oxygen consumption rate (OCR) and extracellular acidification rate (ECAR) were monitored using Vero cells infected with RVPs, with the XFe extracellular flux analyzer (Agilent Seahorse Technologies, Santa Clara, CA, USA). Briefly, 10,000 Vero cells/well were plated into XF96 microplates for ECAR measurement, and 15,000 cells/well for OCR measurement. Prior to the analysis, media was changed to XF DMEM (Agilent Seahorse Technologies, Santa Clara, CA, USA) supplemented with 10 mM glucose (Agilent Seahorse Bioscience), 1 mM sodium pyruvate and 2 mM L-glutamine for OCR (readout of mitochondrial respiration) and to XF DMEM (Agilent Seahorse Technologies, Santa Clara, CA, USA) supplemented with 2 mM of glutamine for ECAR monitorization as an indirect readout of acidification by the production of lactate (aerobic glycolysis). For ECAR measurement the following sequential injections were performed: glucose (10 mM), oligomycin (2 µM), and 2-DG (75 mM). For OCR, sequential injection of oligomycin (2 µM), trifluoromethoxy carbonylcyanide phenylhydrazone (FCCP; 1.2 µM), and rotenone/antimycin A (0.5 µM) as previously described [21 (link), 22 (link)].
3
Macrophage Respiratory Activity Quantification
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The respiratory activity of macrophages was quantified using the Seahorse XF Cell Mito Stress Test Kit and XFe24 Extracellular Flux Analyzer (Seahorse Bioscience) according to a previous study with some modifications [28] (link). Briefly, RAW264.7 cells were plated at a density of 1.5 × 105 cells per well and cultured overnight before the start of experiments. The cells were washed with PBS and then pretreated with 0.08, 0.8, or 8 μg mL-1 enrofloxacin and 10 μg mL-1 ciprofloxacin for 12 h in DMEM supplemented with 1% FBS. The Mito Stress Test Kit assay was then performed using 2 μmol L-1 oligomycin, 1 μmol L-1 Carbonyl cyanide-p-trifluoromethoxyphenylhydrazone (FCCP) and 0.5 μmol L-1 rotenone/antimycin A in XF DMEM (Seahorse Bioscience) supplemented with 10 mmol L-1 glucose, 1 mmol L-1 pyruvate and 2 mmol L-1 L-glutamine. The respiratory capacity was computed as the differences between various stages.
4
Mitochondrial Respiration in 3T3-L1 Cells
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Briefly, 3T3-L1 cells were grown and differentiated in XFe96 well plates coated with gelatin. On the assay day, the cell culture medium was changed to Seahorse XF DMEM supplemented with 1 mM pyruvate, 2 mM glutamate, and 10 mM glucose. The plate was incubated in a non-CO2 incubator at 37 °C for 1 h after oligomycin (2 µM), FCCP (2 µM), and rotenone/antimycin (1 µM) were added to their respective ports prior to the assay. After cartridge calibration, the cells were placed in XFe96 analyzer (Agilent Seahorse Bioscience, Santa Clara, CA, USA) to measure the oxygen consumption rate.
5
Measuring Mitochondrial Respiration in K562 Cells
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1.50x105 K562 cells per well were plated on a Seahorse plate coated with Cell-Tak Cell and Tissue Adhesive (Corning Life Sciences) in XF DMEM (Agilent) containing 5.55mM glucose, 4mM Glutamine, 1mM sodium pyruvate, and oxygen consumption was recorded using a Seahorse XF96 Analyzer (Seahorse Biosciences). Each measurement was performed over 6 min after a 3 min mix and a 3 min wait period. Basal measurements were collected 4 times, followed by 4 measurements after addition of oligomycin (final concentration 2μM), followed by 4 measurements after addition of Bam15 (final concentration 3μM), followed by 4 measurements after addition of Piericidin A+ Antimycin A (final concentration 0.5 μM).
6
Extracellular Flux Analysis of Cellular Metabolism
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Metabolic analysis was performed as a real-time measurement through extracellular flux analysis with XFp extracellular flux analyzer (Agilent Seahorse Technologies, Santa Clara, CA, USA). The oxygen consumption rate (OCR) and extracellular acidification rate (ECAR) were measured with XFp flux analyzer through application of the cell energy phenotype test kit (Agilent Seahorse Technologies) according to the manufacturer’s instructions. As metabolic inhibitors, the ATP synthase inhibitor oligomycin (1 µM) was used as a co-injection with trifluoromethoxy carbonylcyanide phenylhydrazone (FCCP), (1 µM) after 3 basal measurement points. BeWo were plated in XFp miniplates at a density of 0.5 to 1 × 104 cells per well. Measurements were performed in biological and experimental triplicates. A medium change prior to measurement was conducted with XF DMEM (#103575-100, Agilent Seahorse Technologies) under the addition of 10 mM glucose, 1 mM sodium pyruvate and 2 mM L-glutamine. Normalization of OCR and ECAR values was performed through total protein content determined as optical density (OD) by Bradford assay.
7
Cellular Metabolic Profiling with Seahorse
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Cells were dissociated with Accutase and then suspended in high‐glucose DMEM. Cells were then seeded in an Agilent Seahorse XF24 cell culture microplate (Agilent Technologies, Santa Clara, CA, USA; 6 × 104 cells per well), and the plates were incubated for 24 h. The culture medium was replaced with an analysis medium, high‐ or low‐glucose XF DMEM (Cat#103575‐100, Agilent Technologies), and the oxygen consumption rate (OCR) and extracellular acidification rate (ECAR) were analyzed using an Agilent Seahorse XFe24 analyzer (Agilent Technologies). The analysis medium contained 2 mm Seahorse XF glutamine solution (Agilent Technologies), 1 mm Seahorse XF pyruvate solution (Agilent Technologies), and 25 mm glucose in the high‐glucose medium or 5.5 mm glucose in the low‐glucose medium. The OCR and ECAR were analyzed at two time points, 1 h (short period) and 24 h (long period), after replacement with fresh high‐ and low‐glucose DMEM. Additionally, the rate of ATP production was analyzed using a Seahorse XF Real‐Time ATP Rate Assay Kit (Cat# 103592‐100, Agilent Technologies), according to the manufacturer's protocol.
8
Oxygen Consumption Rate Analysis of Live ECs
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For oxygen consumption rate (OCR) analysis of live ECs were analyzed using an XF-96 Extracellular Flux Analyzer (Agilent Technologies). Cells were plated in XF-96 cell culture plates 5∗10ˆ4 cells/cm2 and treated with or without doxycycline (Sigma-Aldrich, D3447) for 24h. After treatment, cells were washed and incubated for 45min in a non-CO2 incubator with XF DMEM (Agilent Technologies) supplemented with 1 mM pyruvate, 2 mM glutamine and 10 mM glucose. Following incubation OCR was analyzed under basal conditions and after to 2 μM oligomycin, 200 μM 2,4-dinitrofenol (DNP) and 1 μM rotenone plus 1 μM antimycin A. Analysis was performed utilizing Seahorse Wave 2.6.1 Desktop Software (Agilent)
9
Cell Culture Reagents and Procedures
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RPMI 1640 culture media, penicillin/streptomycin, fetal bovine serum (FBS), phosphate buffered saline (PBS) and BCA Protein Assay Kit were all obtained from Thermo Fisher Scientific (Waltham, MA, USA). XF DMEM and XF-PS 96-well plates were obtained from Agilent Technologies (Santa Clara, CA, USA). The RNeasy mini kit was obtained from Qiagen (Hilden, Germany) and the High Capacity cDNA Reverse Transcription Kit and Power SYBR Green PCR Master Mix were procured from Applied Biosystems (Waltham, MA, USA). All other chemicals were obtained from Sigma-Aldrich (St. Louis, MO, USA).
10
Assessing Respiration in Mouse Adipose Tissue
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For assessing respiration in iWAT and BAT, the protocol by Bugge et al (2014 (link)) was followed with minor modifications. Briefly, freshly isolated mouse BAT and iWAT pieces were rinsed with XF-DMEM (Agilent) supplemented with 25 mM HEPES. The tissues were thoroughly cleaned of non-adipose material and blood vessels and cut into ~10 mg pieces. After repeated rinses, every piece was placed in the center of a well of XF24 Islet Capture microplate (#101122-100 Seahorse, Agilent) and then covered with the customized screens provided by the manufacturer. Immediately after, XF-DMEM, supplemented with 1 mM sodium pyruvate, 25 mM glucose and 1 mM glutamine, was added to each well. Then OCRs were analyzed with a Seahorse XFe24 Extracellular Flux Analyzer (Agilent, Santa Clara, CA, USA). To stimulate mitochondrial respiration in iWAT, carbonyl cyanide 4-(trifluoromethoxy) phenylhydrazone (FCCP; Sigma-Aldrich, St. Louis, MO), antimycin A (Sigma-Aldrich, St. Louis, MO) and rotenone (Sigma-Aldrich, St. Louis, MO) were injected at specific time points at a final concentration of 20 µM for each compound.
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