Xf rpmi medium

Manufactured by Agilent Technologies

Sourced in United States

XF RPMI medium is a specialized cell culture medium developed by Agilent Technologies. It is designed to support the optimal growth and metabolism of cells in extracellular flux analysis experiments.

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15 protocols using xf rpmi medium

Mitochondrial Respiration Profiling

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ORC were measured using a Seahorse XF Cell Mito Stress Test Kit (Agilent Technologies) according to the manufacturer’s instructions. Briefly, MonoMac6 (0.06 million cells/well) or Molm13 (0.1 million cells/well) cells were washed with XF RPMI medium (Agilent Technologies) supplemented with 1 mM pyruvate, 2 mM glutamine, and 10 mM glucose, seeded in XF96 Cell Culture Microplate coated with Cell-Tak (Corning), and incubated in XF RPMI medium (Agilent Technologies) supplemented with 1 mM pyruvate, 2 mM glutamine, and 10 mM glucose for 30 min to 45 min at 37 °C (non-CO₂ incubator). OCR was determined in the presence of the mitochondrial inhibitor oligomycin (1.5 μM), mitochondrial uncoupling compound carbonyl cyanide 4-(trifluoromethoxy)phenylhydrazone (FCCP) (0.5 μM for MonoMac6 cells and 1.0 μM for Molm13 cells), and respiratory chain inhibitor Rotenone & antimycin A mixture (Rot/AA) (0.5 μM).

Weng H., Huang F., Yu Z., Chen Z., Prince E., Kang Y., Zhou K., Li W., Hu J., Fu C., Aziz T., Li H., Li J., Yang Y., Han L., Zhang S., Ma Y., Sun M., Wu H., Zhang Z., Wunderlich M., Robinson S., Braas D., Hoeve J.T., Zhang B., Marcucci G., Mulloy J.C., Zhou K., Tao H.F., Deng X., Horne D., Wei M., Huang H, & Chen J. (2022). The m6A Reader IGF2BP2 Regulates Glutamine Metabolism and Represents a Therapeutic Target in Acute Myeloid Leukemia. Cancer cell, 40(12), 1566-1582.e10.

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Mitochondrial Function Analysis in PSC-CMs

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Mitochondria function was analyzed with Seahorse XF96 extracellular flux analyzer. Seahorse XF96 microplate was coated with 0.1% gelatin or ECMs. At day 10 of cardiac differentiation, the PSC-CMs were seeded onto the plate with a density of 50,000 cells/well. The cells were cultured until day 38 before starting the assay. The culture medium was changed for base medium (Seahorse XF RPMI medium supplemented with 1 mM pyruvate, 2 mM glutamine, and 25 mM glucose) for 1 hour before running assay and during measurement. Selective inhibitors were sequentially injected during the measurements at the final concentrations of 3 μM oligomycin (an inhibitor for complex V [ATPase] of mitochondrial electron transport chain [METC]), 0.5 μM carbonyl cyanide-p-trifluoromethoxyphenylhydrazone (FCCP, as a mitochondrial oxidative phosphorylation uncoupler), and 3 μM rotenone (an inhibitor for complex I of METC) and antimycin A (an inhibitor for complex III of METC). Basal respiration was represented by oxygen consumption rate (OCR) before applying oligomycin. ATP-linked respiration was represented by the oligomycin-sensitivity respiration rate, while proton leak was calculated by the difference between oligomycin and rotenone/antimycin A rates. Maximal mitochondrial respiration was the response to FCCP.

Chanthra N., Abe T., Miyamoto M., Sekiguchi K., Kwon C., Hanazono Y, & Uosaki H. (2020). A Novel Fluorescent Reporter System Identifies Laminin-511/521 as Potent Regulators of Cardiomyocyte Maturation. Scientific Reports, 10, 4249.

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Real-Time Macrophage Metabolism Analysis

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To track glycolysis and oxidative metabolism during activation of macrophages in real time, 1 × 10⁵ macrophages/well were plated in XF96 V3 cell culture plates and cultured overnight. The cell culture medium was replaced with 180 μl Seahorse XF RPMI medium with 10 mM D-glucose and 4 mM L-glutamine (pH 7.4). After incubation without CO₂ for 45 min, OCR and ECAR were recorded at basal level and for at least 4 h after addition of 20 or 50 μg/ml LPS, 0.5 mg/ml cAMP, or medium for unstimulated controls as the first injection in the Seahorse run. The standard 20 min equilibration cycle at the beginning of a Seahorse run was replaced by an incubation for 10 min without additional mixing. Measurement cycles consist of 30 s mixing, 1.5 min waiting, and 3 min measuring. A minimum of four technical replicates were used for each condition.

Wentzel A.S., Janssen J.J., de Boer V.C., van Veen W.G., Forlenza M, & Wiegertjes G.F. (2020). Fish Macrophages Show Distinct Metabolic Signatures Upon Polarization. Frontiers in Immunology, 11, 152.

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Bioenergetic Profiling of CD4+ T Cells

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The bioenergetic activity of CD4⁺ T cells was measured using the Seahorse XFe96 Analyzer. Magnetically enriched T cells were seeded at 2 × 10⁵ cells/well. Cells were seeded on a Cell-Tak (Corning #354240)-coated XFe96 plate with fresh XF media (Seahorse XF RPMI medium with 2 mM glutamine, 10 mM glucose, 1 mM sodium pyruvate, and 5 mM HEPES (4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid), pH 7.4). For the Mito Stress Assay, OCR was measured with additions of oligomycin (1.5 μM), FCCP (2-[2-[4-(trifluoromethoxy)phenyl]hydrazinylidene]-propanedinitrile, 1.5 μM), rotenone (1 μM), and antimycin A (1 μM) during the assay.

Wilfahrt D., Philips R.L., Lama J., Kizerwetter M., Shapiro M.J., McCue S.A., Kennedy M.M., Rajcula M.J., Zeng H, & Shapiro V.S. (2021). Histone deacetylase 3 represses cholesterol efflux during CD4+ T-cell activation. eLife, 10, e70978.

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Hypoxic CD8+ T Cell Metabolism

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Mouse CD8⁺ T cells activated for 3 days in 1% O₂ were assayed in a Seahorse Extracellular Flux Analyzer XF96 (Agilent) to determine oxygen consumption rate (OCR) and extracellular acidification rate (ECAR). The Seahorse assay was conducted in a hypoxia chamber at 3% O₂. 1.5x10⁵ CD8⁺ T cells were plated onto poly-D-lysine-coated wells in XF RPMI medium (Agilent), pH 7.4, supplemented with 10 mM glucose (ThermoFisher) and 2 mM glutamine (ThermoFisher). Media was pre-incubated at 1% O₂. A minimum of 5 technical replicates per biological replicate were used. During the assay, wells were sequentially injected with anti-CD3/CD28 dynabeads (4:1 bead to T-cell ratio), 1 μM oligomycin (Sigma), 1.5 μM FCCP (Sigma), and 100 nM rotenone (Sigma) + 1 μM antimycin A (Sigma).

Cunha P.P., Bargiela D., Minogue E., Krause L.C., Barbieri L., Brombach C., Gojkovic M., Marklund E., Pietsch S., Foskolou I., Branco C.M., Veliça P, & Johnson R.S. (2022). Infiltration of Tumors Is Regulated by T cell–Intrinsic Nitric Oxide Synthesis. Cancer Immunology Research, 11(3), 351-363.

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Metabolic Profiling of Splenic Treg Cells

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The rates of mitochondrial oxygen consumption and glycolysis were measured using the Seahorse XF-96 analyzer (Agilent). Splenic T_reg cells (YFP⁺) (250,000) were isolated by flow sorting and adhered to XF96 cell culture plates using Cell-Tak (Corning) per the manufacturer’s instructions. Cells were plated in XF RPMI medium (Agilent) supplemented with 1% FBS, 11 mM glucose, 2 mM glutamine, and 1 mM pyruvate to match normal concentrations of those metabolites in base RPMI. The basal mitochondrial OCR was determined by subtracting the Antimycin A– and piericidin A–sensitive oxygen consumption from the basal mitochondrial oxygen consumption. The basal glycolytic rate was calculated by subtracting the 2-Deoxyglucose–sensitive ECAR from the basal ECAR. Antimycin A and piericidin A purchased from Sigma-Aldrich were both used at a final concentration of 1 μM. 2-Deoxyglucose purchased from Sigma-Aldrich was used at a final concentration of 25 mM.

Montauti E., Weinberg S.E., Chu P., Chaudhuri S., Mani N.L., Iyer R., Zhou Y., Zhang Y., Liu C., Xin C., Gregory S., Wei J., Zhang Y., Chen W., Sun Z., Yan M, & Fang D. (2022). A deubiquitination module essential for Treg fitness in the tumor microenvironment. Science Advances, 8(47), eabo4116.

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Measuring Macrophage Metabolic Parameters

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For measuring the metabolic parameters with the Seahorse bioanalyzer device, primary murine macrophages were isolated and differentiated as previously described (part 2.15). The macrophages were harvested in the same way as described in the part 2.17, seeded (25 × 10³ cells/well) into Seahorse 96-well cell culture plates and incubated overnight. On the day of measurement, the media was replaced by XF RPMI medium supplemented with 10 mM glucose and 2 mM glutamine (all from Agilent). The plate was equilibrated for 30 min in a non-CO₂ incubator at 37 °C. Metabolic parameters were measured on a Seahorse XFe 96 extracellular flux analyzer (Agilent). To analyze the ATP production rate, 2.5 µM oligomycin and 500 nM rotenone together with antimycin A were sequentially added to the cells. All chemicals were purchased from Cayman chemicals. Data were processed using Wave Desktop (Version 2.6.0.31) and ATP rates were calculated using the Seahorse Analytics online tool (Version from February 2022).

Wedel S., Mathoor P., Rauh O., Heymann T., Ciotu C.I., Fuhrmann D.C., Fischer M.J., Weigert A., de Bruin N., Hausch F., Geisslinger G, & Sisignano M. (2022). SAFit2 reduces neuroinflammation and ameliorates nerve injury-induced neuropathic pain. Journal of Neuroinflammation, 19, 254.

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Measuring Metabolic Profiles of Activated CD8+ T Cells

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Purified CD8+ T cells were activated for 12 hours, then 3x10⁵ CD8+ T cells were plated onto poly-D-lysine coated wells in order to form a homogeneous monolayer. Cells were assayed in XF RPMI medium (Agilent) pH 7.4 supplemented with 2 mM glutamine. A minimum of 4 technical replicates per each biological replicate was used. Oxygen consumption rate (OCR) and extracellular acidification rate (ECAR) were determined through a Seahorse Extracellular Flux Analyzer XF96 (Agilent). During the assay, wells were sequentially injected with 40 mM NaCl/NaLac with or without the MCT1-selective inhibitor AZD3965 (Cayman Chemical, 19912) or the LDH inhibitor GSK2837808A (Bio-Techne, 5189), or plain media alone, followed by 10 mM glucose (Sigma), 1 µM oligomycin and 50 mM 2-deoxyglucose (2-DG; Sigma D6134).

Barbieri L., Veliça P., Gameiro P.A., Cunha P.P., Foskolou I.P., Rullman E., Bargiela D., Johnson R.S, & Rundqvist H. (2023). Lactate exposure shapes the metabolic and transcriptomic profile of CD8+ T cells. Frontiers in Immunology, 14, 1101433.

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Quantifying Cellular Energy Metabolism

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Real-time analysis of energy metabolism was performed by using a flux analyzer (Seahorse Bioscience XF24 Extracellular Flux Analyzer; Agilent Technologies, Santa Clara, CA, USA) and XF Mito Stress Kit (Agilent Technologies) according to the manufacturers’ protocols. Briefly, the cell culture microplate (Agilent Technologies) was treated with 16.5 μg/mL Cell-TAK (Corning) in 0.1 mol/L NaOH and incubated at room temperature for 25 min. The plate was washed with distilled water after removing Cell-TAK liquid, and cells were cultured at 8×10⁴ cells/well with XF RPMI Medium (Agilent Technologies) containing glutamine (2 mmol/L, Agilent Technologies), glucose (10 mmol/L, Agilent Technologies), and pyruvate (1 mmol/L, Agilent Technologies) until starting flux analysis. The plate (Agilent Technologies) was set into the flux analyzer, and the following compounds (final concentrations) were injected during the assay: 1.5 µM oligomycin (inhibitor of ATP synthase), 1.5 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 Technologies) was used to analyze the results.

Ishida K., Nagatake T., Saika A., Kawai S., Node E., Hosomi K, & Kunisawa J. (2023). Induction of unique macrophage subset by simultaneous stimulation with LPS and IL-4. Frontiers in Immunology, 14, 1111729.

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Hypoxic CD8+ T Cell Metabolism

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Mouse CD8⁺ T cells activated for 3 days in 1% O₂ were assayed in a Seahorse Extracellular Flux Analyzer XF96 (Agilent) to determine oxygen consumption rate (OCR) and extracellular acidification rate (ECAR). The Seahorse assay was conducted in a hypoxia chamber at 3% O₂. 1.5×10⁵ CD8⁺ T cells were plated onto poly-D-lysine-coated wells in XF RPMI medium (Agilent), pH 7.4, supplemented with 10 mmol/L glucose (ThermoFisher) and 2 mmol/L glutamine (ThermoFisher). Media was preincubated at 1% O₂. A minimum of 5 technical replicates per biological replicate were used. During the assay, wells were sequentially injected with anti-CD3/CD28 Dynabeads (4:1 bead to T-cell ratio), 1 μmol/L oligomycin (Sigma), 1.5 μmol/L FCCP (Sigma), and 100 nmol/L rotenone (Sigma) + 1 μmol/L antimycin A (Sigma).

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