Xfe96 analyzer

Manufactured by Agilent Technologies

Sourced in United States

The XFe96 Analyzer is a high-performance metabolic analysis system designed for cell-based assays. It measures the oxygen consumption rate (OCR) and extracellular acidification rate (ECAR) of cells, providing insights into cellular metabolism and function. The analyzer uses advanced fluorescence detection technology to enable real-time, non-invasive monitoring of cellular respiration and glycolysis.

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198 protocols using xfe96 analyzer

Metabolic Profiling of CPT-11-Treated T Cells

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Seahorse XFe96 Analyzers was used to measure the extracellular acidification rate (ECAR) and oxygen consumption rate (OCR) of CD4⁺ T cells treated with CPT-11 (10 μg/mL) or PBS for 24 h. 2 × 10⁵ cultured T cells, resuspended in XF RPMI medium supplemented with 10 mM glucose, 1 mM pyruvate and 2 mM L-glutamine, were added into Seahorse XF96 cell culture microplates (coated with 22.4 μg/mL Corning® Cell-Tak™ Cell and Tissue Adhesive). In the absence of CO₂, cells in microplates were incubated with shaking at 37 °C for approximately 30 min, and, subsequently, the Seahorse XF Cell Mito Stress Test program was run. To measure basal glycolysis in T cells treated with CPT-11 or PBS, the indicated cells were harvested after 24 h. Next, the cells were resuspended in the aforementioned XF RPMI medium and incubated with CPT-11 or PBS, and basal ECAR was measured.

Liang H., Fan X., Cheng H., Ma X., Sun Y., Nan F., Zhou J., Shu P., Zhang W., Zuo F., Nakatsukasa H, & Zhang D. (2024). CPT-11 mitigates autoimmune diseases by suppressing effector T cells without affecting long-term anti-tumor immunity. Cell Death Discovery, 10, 218.

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Metabolic Profiling of CD4+ T Cells

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Extracellular acidification rate (ECAR) and oxygen consumption rate (OCR) of CD4+ T cells were measured using a Seahorse XFe96 Analyzers. T cells were cultured with or without BMP4 (10ng/ml, PeproTech) for 24 hours, and then were prepared in XF RPMI medium supplemented with 10 mM glucose, 1 mM pyruvate and 2 mM L-glutamine, added 2 × 10⁵ T cells into each well of Seahorse XF96 cell culture microplates (Coated with 22.4 μg/ml Corning^® Cell-Tak Cell and Tissue Adhesive), spined down and preincubated at 37°C for around 30 min in the absence of CO², then ran the program of the Seahorse XF Cell Mito Stress Test.

Huang F., Hu L., Zhang Y., Qu X, & Xu J. (2021). BMP4 Moderates Glycolysis and Regulates Activation and Interferon-Gamma Production in CD4+ T Cells. Frontiers in Immunology, 12, 702211.

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Metabolic Profiling of CD4+ T Cells

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Extracellular acidification rate (ECAR) and oxygen consumption rate (OCR) of CD4⁺ T cells were measured using a Seahorse XFe96 Analyzers. For common tests, prepared cultured cells or in vivo isolated cells in XF RPMI medium supplemented with 10 mM glucose, 1 mM pyruvate and 2 mM L-glutamine, added 2 × 10⁵ (in vitro cultured for 24 h) or 3 × 10⁵ (fresh isolated from in vivo) T cells into each well of Seahorse XF96 cell culture microplates (Coated with 22.4 μg/ml Corning® Cell-Tak™ Cell and Tissue Adhesive), spined down and preincubated at 37°C for around 30 min in the absence of CO₂, then ran the program of the Seahorse XF Cell Mito Stress Test. For the measurement of basal glycolysis of T cells cultured with 5.5 mM, 25 mM and 50 mM glucose, cells were harvested after 24 h culture in indicate concentrations of glucose or galactose, then prepared the cells in XF RPMI medium supplemented with 1 mM pyruvate, 2 mM L-glutamine and indicate concentrations of glucose or galactose (Same concentrations with T cell culture), then preincubated and measured the basal ECAR.

Zhang D., Jin W., Wu R., Li J., Park S.A., Tu E., Zanvit P., Xu J., Liu O., Cain A, & Chen W. (2019). High glucose intake exacerbates autoimmunity through reactive oxygen species-mediated TGF-β cytokine activation. Immunity, 51(4), 671-681.e5.

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Glycolytic Profiling of MAOA-Overexpressing Cancer Cells

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Agilent Seahorse XFe96 Analyzers (Beijing, China) were used to measure the extracellular acidification rate (ECAR) of cancer cells in a 96-well-plate followed the manufacturer's manual.
The glucose consumption and lactate production in stable MAOA overexpressing cells and the corresponding control cells were detected as follows. Cells were seeded into 35-mm dishes for 36 h. The supernatants of cell culture medium were collected by centrifugation at 800 rpm for 5 min. The Glucose Assay Kit (Sigma, Shanghai, China) and the Lactate Assay kit (BioVision, Milpitas, CA, USA) were used to determine the level of glucose and lactate, respectively. The PicoProbe™ Hexokinase Activity Assay Kit (BioVision, CA, USA) was used to detect HK2 activity, as previously reported (5 (link)).

Huang Y., Zhao W., Ouyang X., Wu F., Tao Y, & Shi M. (2021). Monoamine Oxidase A Inhibits Lung Adenocarcinoma Cell Proliferation by Abrogating Aerobic Glycolysis. Frontiers in Oncology, 11, 645821.

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Quantifying Mitochondrial ATP Production

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Percent ATP produced by mitochondria and the level of compensatory glycolysis were measured using a Seahorse XF Real-Time ATP Rate Assay Kit and XFe96 Analyzers (Agilent) according to manufacturer instructions. Seahorse XF Analyzers directly measure real time extracellular acidification rate (ECAR) and oxygen consumption rate (OCR) of cells. ECAR and OCR are indicators of the two major energy-producing pathways: glycolysis and oxidative phosphorylation. Basal OCR and ECAR rates were first measured. Injection of oligomycin results in an inhibition of mitochondrial ATP synthesis that results in a decrease in OCR, allowing for quantification of mitochondrial ATP Production Rate. ECAR data combined with the buffer factor of the assay medium allows calculation of total Proton Efflux Rate (PER) using the following formula: PER (pmol H + /min) = ECAR (mpH/min) × BF (mmol/L/pH) × Geometric Volume (μL) × Kvol. Complete inhibition of mitochondrial respiration with rotenone plus antimycin A accounts for mitochondrial-associated acidification, and when combined with PER data allows calculation of the glycolytic ATP Production Rate. Compensatory glycolysis was measured as ATP production with complete inhibition of mitochondrial respiration.

Przanowski P., Przanowska R.K, & Guertin M.J. (2023). ANKLE1 cleaves mitochondrial DNA and contributes to cancer risk by promoting apoptosis resistance and metabolic dysregulation. Communications Biology, 6, 231.

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Metabolic Analysis of NK Cells

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NK cells were enriched from FL and 1αKO mice and 4 × 10⁵ cells were transferred into 96-well plate without any stimulation. Agilent Seahorse XF Cell Mito Stress test was performed on NK cells using oligomycin (10 μM), FCCP (10 μM), rotenone (10 μM) plus antimycin A (10 μM) over 2 hr. Oxygen consumption rate (OCR) and extracellular acidification rate (ECAR) was measured and analyzed using Agilent Seahorse XFe96 Analyzers.

Victorino F., Bigley T.M., Park E., Yao C.H., Benoit J., Yang L.P., Piersma S.J., Lauron E.J., Davidson R.M., Patti G.J, & Yokoyama W.M. (2021). HIF1α is required for NK cell metabolic adaptation during virus infection. eLife, 10, e68484.

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Seahorse Assay of NK Cells

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NK cells were enriched from FL and 1α KO mice and 4x10 5 cells were transferred into 96 well plate without any stimulation. Agilent Seahorse XF Cell Mito Stress test was performed on NK cells using oligomycin (10 μM), FCCP (10 μM), rotenone (10 μM) plus antimycin A (10 μM) over two hours. Oxygen consumption rate (OCR) and extracelluar acidification rate (ECAR) was measured analyzed using Agilent Seahorse XFe96 Analyzers.

Victorino F., Bigley T.M., Park E., Yao C., Benoit J., Yang L., Piersma S.J., Lauron E.J., Davidson R.M., Patti G.J., & Yokoyama W.M. (2021). NK cell metabolic adaptation to infection promotes survival and viral clearance.

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Metabolic Profiling of Activated PBMCs

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Metabolic profile was evaluated in PBMCs stimulated for 12 h in the absence or presence of anti-CD3 (clone OKT3). ECAR and OCR were measured with an XFe-96 Analyzer (Seahorse Bioscience). Specifically, cells were plated in XFe-96 plates (Seahorse Bioscience) at a concentration of 4 × 10⁵ cells per well and cultured with RPMI-1640 medium supplemented with 5% autologous plasma. ECAR was measured in XF media (nonbuffered Dulbecco’s modified Eagle’s medium containing 10 mM glucose and 1 mM sodium pyruvate) under basal conditions and in response to 10 mM glucose, 5 μM oligomycin, and 100 mM 2-deoxy-d-glucose (all from Sigma–Aldrich). OCR was measured in XF media under basal conditions and in response to 5 μM oligomycin, 1.5 μM carbonylcyanide-4-(trifluoromethoxy)-phenylhydrazone, and 1 μM antimycin A and rotenone (Sigma–Aldrich). Experiments with the Seahorse were done with the following assay conditions: 3-min mixture, 3-min wait, and 3-min measurement. ECAR was also measured on T_conv cells stimulated with anti-CD3/CD28 (0.2 bead per cell) in the presence or not of hrLeptin (300 ng/mL) at indicated time points.

Bruzzaniti S., Bocchino M., Santopaolo M., Calì G., Stanziola A.A., D’Amato M., Esposito A., Barra E., Garziano F., Micillo T., Zuchegna C., Romano A., De Simone S., Zuccarelli B., Mottola M., De Rosa V., Porcellini A., Perna F., Matarese G, & Galgani M. (2019). An immunometabolic pathomechanism for chronic obstructive pulmonary disease. Proceedings of the National Academy of Sciences of the United States of America, 116(31), 15625-15634.

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Mitochondrial Respiration Assay in CMs

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Respiration rates were measured with Seahorse XFe96 Analyzer. CMs were plated at 1.5 × 10⁴ cells per well of a 96 well XF96 Cell Culture Microplate (Aligent Technologies) and cultured for 3 days. One hour before the assay, the medium was changed to RPMI without phenol red supplemented with sodium pyruvate. The Seahorse Extracellular Flux Assay Kit was used with the Mito Stress Test protocol. Inhibitor final concentrations were Oligomyocin (2.5 μM), FCCP (1 μM), and Rotenone (2.5 μM) + Antimycin A (2.5μM).

Murphy S.A., Miyamoto M., Kervadec A., Kannan S., Tampakakis E., Kambhampati S., Lin B.L., Paek S., Andersen P., Lee D.I., Zhu R., An S.S., Kass D.A., Uosaki H., Colas A.R, & Kwon C. (2021). PGC1/PPAR drive cardiomyocyte maturation at single cell level via YAP1 and SF3B2. Nature Communications, 12, 1648.

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TNF-α Receptor Modulation and Cell Viability

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HT-22 cells were pre-treated for 1 h with 4, 6, or 8 μg/mL of LEAF™ Purified anti-mouse CD120a (TNF R Type I/p55), 4, 6, or 8 μg/mL of LEAF™ Purified anti-mouse CD120b (TNF R Type II/ p75), or 8 μg/mL of LEAF™ Purified Armenian Hamster IgG Isotype Ctrl from Biolegend. Media was removed and cells were then treated with 100 pg/mL of TNF-α for 1.5 h or 3 h. Cell viability was assessed with Calcein AM at both 1.5 and 3 h, and mitochondrial basal respiration was assessed with the XF^e 96 Analyzer from Seahorse Bioscience at 1.5 h.

Doll D.N., Rellick S.L., Barr T.L., Ren X, & Simpkins J.W. (2015). Rapid mitochondrial dysfunction mediates TNF-alpha-induced neurotoxicity. Journal of neurochemistry, 132(4), 443-451.

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