Xf base medium

Manufactured by Agilent Technologies

Sourced in United States, Canada

XF base medium is a specialized cell culture medium designed for use with Agilent's Seahorse XF Analyzers. It provides a buffered solution to maintain optimal conditions for cells during metabolic flux analysis.

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Seahorse XF Base Medium (107 citations) Agilent Seahorse XF Base Medium (17 citations) Base medium (16 citations) XF Base Medium Minimal DMEM (12 citations) XF DMEM base medium (11 citations) Seahorse XF base medium pH7.4 (5 citations) Seahorse XF Base Medium Minimal DMEM (4 citations) Seahorse XF base medium without phenol red (4 citations) XF Base Medium (minimal Dulbecco’s Modified Eagle’s Medium) (2 citations) Seahorse XF DMEM base medium (2 citations)

166 protocols using xf base medium

Metabolic Profiling of A549 and PC9 Cells

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A549 and PC9 cells were plated at 7500 cells/well in XF96 microplates (Seahorse Bioscience, Billerica, MA, USA) in RPMI medium with 10% according to the manufacturer's recommendations 48 h prior to the assay. Treatment with 1.5 mM LA was performed for 24 h.
For Mito Stress Test, the medium was exchanged to XF base medium (Seahorse Bioscience, Billerica, MA, USA) with 1 mM pyruvate, 2 mM glutamine, and 10 mM glucose and incubated at 37°C without CO₂ 1 h prior to the assay. During the assay, OCR was measured using a Seahorse XFe 96 analyzer. Mito Stress Test kit containing 1 μM oligomycin, 1 μM carbonyl cyanide-4-(trifluoromethoxy)phenylhydrazone (FCCP), and 0.5 μM rotenone/0.5 μM antimycin A was performed according to the manufacturer's protocol.
For glycolytic rate assay, the medium was exchanged to XF base medium without phenol red (Seahorse Bioscience, Billerica, MA, USA) with 2 mM glutamine, 10 mM glucose, 1 mM pyruvate, and 5 mM HEPES and incubated at 37°C without CO₂ 1 h prior to the assay. During the assay, ECAR was measured using a Seahorse XFe 96 analyzer. Glycolytic rate assay kit containing 0.5 μM rotenone, 0.5 μM antimycin A, and 50 mM 2-desoxyglucose was performed according to the manufacturer's protocol. ECAR and OCR were calculated by Wave 2.6 (Seahorse Bioscience, Billerica, MA, USA) software after the assay and normalized to cell numbers.

Yue L., Ren Y., Yue Q., Ding Z., Wang K., Zheng T., Chen G., Chen X., Li M, & Fan L. (2021). α-Lipoic Acid Targeting PDK1/NRF2 Axis Contributes to the Apoptosis Effect of Lung Cancer Cells. Oxidative Medicine and Cellular Longevity, 2021, 6633419.

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Bioenergetic Profiling of Infected Macrophages

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Bioenergetic profile of infected macrophages was measured by determining oxygen consumption rate (OCR) and extracellular acidification rate (ECAR) in a XF-24 Flux Analyzer (Seahorse Bioscience). Sensor cartridges (Agilent Technologies) were hydrated in XF Calibrant (Agilent Technologies) at 37°C overnight following manufacturer’s instructions. Media for Glycolysis stress assay: XF Base Medium (Seahorse Bioscience) with 2mM glutamine, 1mM pyruvate, 10mM glucose, 5mM HEPES and MitoStress assay: XF Base Medium with 2mM glutamine, 1mM pyruvate and 10mM glucose were made fresh and kept at pH 7.4. Experimental plates were kept at 37°C without CO₂ for 1 hour before loading into the Seahorse machine. Cells were sequentially treated with 10 mM glucose, 1μM oligomycin (O) and 50 mM 2-deoxy-glucose (2DG) to determine glycolysis parameters from the ECAR levels, or with 1 μM O, 1 μM FCCP (carbonyl cyanide-4-(trifluoromethoxy) phenylhydrazone), and 0.5 μM rotenone (ROT) plus antimycin A (AA) to asses OXPHOX parameters from the OCR levels.

Ty M.C., Loke P., Alberola J., Rodriguez A, & Rodriguez-Cortes A. (2019). Immuno-metabolic profile of human macrophages after Leishmania and Trypanosoma cruzi infection. PLoS ONE, 14(12), e0225588.

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Osteocyte Respiration and Glycolysis Profiling

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Primary osteocytes (0.4 × 10⁶ cells/mL, a pool of 2 to 3 mice per group) were seeded on a collagen-coated plate (Cat# 102342–100, Agilent Technologies, Santa Clara, CA, USA) in triplicates. Oxygen consumption rate (OCR) was determined in assay medium (XF base medium Cat# 103193100 supplemented with 1 mM pyruvate, 10 mM glucose, and 2 mM glutamine, Agilent Technologies) using the cellular respiration analyzer (Seahorse Analyzer, Agilent Technologies) at basal, upon oligomycin (1 μM), FCCP (2 μM), and Rotenone/antimycin (0.5 μM) additions. For glycolysis, assay osteocytes were seeded as above using a glucose-free medium (XF base medium Cat# 103193100, Agilent Technologies) with 2 mM glutamine, and glucose (10 mM) added at the indicated time point according to the manufacturer’s instructions. Data were normalized to cell number determined at the end of the assay and analyzed using the Seahorse Wave software. Assays were repeated >4 times using osteocyte preparations from several mice per group. Data from 3 to 6 assays were combined after normalization of basal OCR to 100%.

Liu Z., Solesio M.E., Schaffler M.B., Frikha-Benayed D., Rosen C.J., Werner H., Kopchick J.J., Pavlov E.V., Abramov A.Y, & Yakar S. (2018). Mitochondrial Function Is Compromised in Cortical Bone Osteocytes of Long-Lived Growth Hormone Receptor Null Mice. Journal of bone and mineral research : the official journal of the American Society for Bone and Mineral Research, 34(1), 106-122.

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Seahorse Assay for Glycolysis and Respiration

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Cells were cultured in 24-well Seahorse XFe24 plates (Agilent, Santa Clara, CA) and were incubated at 37 °C under a humidified 5% CO₂ atmosphere. Differentiated mouse myotubes were washed twice per day for 5 days with XF base medium (Agilent) and incubated with XF base medium at 37 °C without CO₂ for 12 h. To determine glycolysis and glycolytic rate, three initial measurements of extracellular acidification rate (ECAR) were made using the XFe24 Seahorse analyzer, and then 10 mM glucose, 4 µg/mL oligomycin, and 100 mM 2DG were sequentially injected and nine additional measurements were performed (three during each condition). To determine oxygen consumption rate (OCR), other cells were initially measured at three time points during basal respiration, and then 4 µg/mL oligomycin (ATP synthase inhibitor), 9 µM carbonyl cyanide 4-(trifluoromethoxy) phenylhydrazone (FCCP; uncoupler of oxidative phosphorylation), and 11 µM antimycin A/5 µM rotenone (blocks mitochondrial respiration) were sequentially injected. During each condition three measurements of OCR were made. Data were analyzed with Wave software (version 2.6.0).

Koh J.H., Pataky M.W., Dasari S., Klaus K.A., Vuckovic I., Ruegsegger G.N., Kumar A.P., Robinson M.M, & Nair K.S. (2022). Enhancement of anaerobic glycolysis – a role of PGC-1α4 in resistance exercise. Nature Communications, 13, 2324.

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Metabolic Profiling of HMEC Cells

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HMEC were cultured at 3 × 10⁴ cells at 24-well Seahorse XF^e24 plates (Agilent) and were incubated at 37 °C under a humidified 5% CO₂ atmosphere overnight. Cells were washed twice with XF base medium (Agilent) and incubated with XF base medium at 37 °C without CO₂ for one hour. Three initial measurements were made using XF^e24 Seahorse analyzer, and then 5 mM glucose, 0.5 mM glutamine, 0.5 mM palmitate, 10 mM lactate, or combinations of them were injected and five additional measurements were performed. Data were analyzed with Wave software (version 2.6.0).

Ocaña M.C., Martínez-Poveda B., Quesada A.R, & Medina M.Á. (2019). Highly Glycolytic Immortalized Human Dermal Microvascular Endothelial Cells are Able to Grow in Glucose-Starved Conditions. Biomolecules, 9(8), 332.

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Fatty Acid Oxidation Assay by Seahorse

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For the FAO-associated oxygen consumption rate (OCR) assay by Seahorse XFp Analyzer (Agilent), Palmitate-BSA reagent (Agilent) and Mito Stress Test Kits (Agilent) were used according to the manufacturers’ instructions with minor modifications. In brief, cells were collected and adhered to poly-D-lysine-coated XFp plates (4 to 6 × 10⁵ cells/well) via centrifugation in serum-free XF Base Medium (Agilent). For evaluation of palmitate oxidation, cells were cultured with XF Base Medium (Agilent) in a non-CO₂ incubator for 15–20 min at 37°C. Subsequently, the XF Palmitate-BSA FAO Substrates (Agilent) palmitate-BSA (167 μM palmitate conjugated with 28 μM BSA) and 0.5 mM L-carnitine (Sigma Aldrich) were added to the medium for the assessment of OCR. Immediately, XF Cell Mito Stress Test was performed in a Seahorse XFp Analyzer (Agilent) by sequentially adding several modulators of mitochondrial function, including 1 mM oligomycin (Oligo) (Agilent), 1.5 mM fluorocarbonyl cyanide phenylhydrazone (FCCP) (Agilent), and 100 nM rotenone plus 1 mM antimycin A (Rot/Ant A) (Agilent). Data were analyzed with Wave software version 2.3.0 (Agilent) and proper statistical methods using GraphPad Prism version 6 (GraphPad Software).

Tian M., Hao F., Jin X., Sun X., Jiang Y., Wang Y., Li D., Chang T., Zou Y., Peng P., Xia C., Liu J., Li Y., Wang P., Feng Y, & Wei M. (2021). ACLY ubiquitination by CUL3-KLHL25 induces the reprogramming of fatty acid metabolism to facilitate iTreg differentiation. eLife, 10, e62394.

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Measuring Mitochondrial Respiration in Cells

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Fibroblasts were seeded at 40,000 cells per well and grown overnight in XF24 plates (Agilent). On the day of the experiment, normal growth medium was replaced by Seahorse XF Base Medium containing 1 mM pyruvate, 2 mM glutamine, and 10 mM glucose, pH 7.4. After 45-min incubation at 37°C without CO₂, the cells were loaded into the Seahorse XF24 Analyzer for calibration. Oxygen consumption rate (OCR) was measured at three time points following serial incubations with oligomycin (final concentration 0.5 μM), FCCP (final concentration 1.0 μM) and antimycin (final concentration 0.5 μM). At the end of the experiment, the cells were lysed with radioimmunoprecipitation assay buffer, and total protein was determined by bicinchoninic acid assay. OCR was normalized to total protein/well. Analysis was performed on five technical replicates and confirmed in two biological replicates.

Zhao T., Goedhart C.M., Sam P.N., Sabouny R., Lingrell S., Cornish A.J., Lamont R.E., Bernier F.P., Sinasac D., Parboosingh J.S., Vance J.E., Claypool S.M., Innes A.M, & Shutt T.E. (2019). PISD is a mitochondrial disease gene causing skeletal dysplasia, cataracts, and white matter changes. Life Science Alliance, 2(2), e201900353.

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Metabolic Profiling of Splenic CD4+ T Cells

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Oxygen consumption rate (OCR, in pmol/min) and extracellular acidification rate (ECAR, in mpH/min) of splenic CD4⁺ T cells were analyzed using a Seahorse XF-24 metabolic extracellular flux analyzer (Seahorse Bioscience, Billerica, MA, USA) as we previously reported [12 (link)]. CD4⁺ T cells were first treated with the indicated reagents for 24 h and then harvested and resuspended in XF Base Medium (Seahorse Bioscience, USA) supplemented with 11 mM glucose, 1 mM pyruvate, and 2 mM glutamine. CD4⁺ T cells were then seeded at a density of 10⁶ cells/well in 24-well XF microplates coated with poly-L-lysine hydrobromide (Sigma Chemical Co., USA). After incubation in a CO₂-free incubator at 37 °C for 1 h, basal metabolism and metabolism in response to mitochondrial inhibitors [1 µM oligomycin, 1 µM FCCP, and 1 µM Rot+1 µM Anti A (Sigma Chemical Co., USA)] were analyzed. The assay conditions were as follows: 3 min of mixing; a 2 min wait; and 3 min of measurement. Metabolic parameters were then calculated. OCR and ECAR were normalized to the cell number in each well.

Lü S.L., Dang G.H., Deng J.C., Liu H.Y., Liu B., Yang J., Ma X.L., Miao Y.T., Jiang C.T., Xu Q.B., Wang X, & Feng J. (2019). Shikonin attenuates hyperhomocysteinemia-induced CD4+ T cell inflammatory activation and atherosclerosis in ApoE−/− mice by metabolic suppression. Acta Pharmacologica Sinica, 41(1), 47-55.

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Bioenergetic Profiling of CEACAM6 Knockout

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CEACAM6 WT and KO HPAF-II cells were seeded in Seahorse cell culture microplates (Seahorse Bioscience #100777-004, 24-well plates), at 40,000 cells/well. Bioenergetic assays (oxygen consumption rates, or OCR) followed Seahorse Bioscience protocols using a machine XFe 24 Extracellular Flux Analyzer (Seahorse Bioscience). To assay oxidation of carbohydrate in the form of added glucose in intact cells, XF Base Medium (Seahorse Bioscience #102353-100) with 10 mM glucose, 2.0 mM glutamine and 1.0 mM pyruvate was used. Other test components included 1.0 µM oligomycin, 1.0 µM FCCP, and 2.0/2.0 µM rotenone/antimycin A. Respiration (OCR) in each well was normalized to cell lysate protein concentration.

Pandey R., Zhou M., Islam S., Chen B., Barker N.K., Langlais P., Srivastava A., Luo M., Cooke L.S., Weterings E, & Mahadevan D. (2019). Carcinoembryonic antigen cell adhesion molecule 6 (CEACAM6) in Pancreatic Ductal Adenocarcinoma (PDA): An integrative analysis of a novel therapeutic target. Scientific Reports, 9, 18347.

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Metabolic Profiling of DU145 Cells

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DU145 mock- and COMP-transfected cells were grown to confluence in a 24-well plate (Seahorse Bioscience) coated with poly-D-lysine (Sigma-Aldrich). The cells were equilibrated in XF Base Medium (Seahorse Bioscience) containing 10 mM glucose for 1.5 hours at 37°C (no CO₂). Oxygen consumption rate (OCR) was measured over time (Seahorse XF24 system, Seahorse Bioscience) as the cells were treated with 25 mM glucose, 1 μg/ml oligomycin A, 0.9 μM FCCP, and finally 1 μM rotenone (Sigma-Aldrich). The cells were lysed and protein content was determined with a BCA kit. The OCR data were normalized to the protein concentration of each well and then to the highest value of each repetition.

Englund E., Canesin G., Papadakos K.S., Vishnu N., Persson E., Reitsma B., Anand A., Jacobsson L., Helczynski L., Mulder H., Bjartell A, & Blom A.M. (2017). Cartilage oligomeric matrix protein promotes prostate cancer progression by enhancing invasion and disrupting intracellular calcium homeostasis. Oncotarget, 8(58), 98298-98311.

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