Seahorse xf96 v3 ps cell culture microplates

Manufactured by Agilent Technologies

Sourced in United Kingdom

The Seahorse XF96 V3 PS Cell Culture Microplates are designed for use with Agilent's Seahorse XF Analyzers. These microplates provide a standardized platform for measuring extracellular acidification and oxygen consumption rates of cells in real-time, allowing researchers to assess cellular metabolic activity.

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6 protocols using seahorse xf96 v3 ps cell culture microplates

Mitochondrial Respiration Profiling

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HAoSMCs were cultivated in 96-well Seahorse XF96 V3 PS Cell Culture Microplates (Agilent Technologies, Waldbronn, Germany). Real-time OCR was determined by Seahorse XF Cell Mito Stress Test Kit and Seahorse XFe96 Analyzer (Agilent Technologies, Waldbronn, Germany) according to the manufacturer’s instructions.
A detailed description of the measurement can be found in Additional file 1 section: supplementary methods.

Terpe P., Ruhs S., Dubourg V., Bucher M, & Gekle M. (2024). The synergism of cytosolic acidosis and reduced NAD+/NADH ratio is responsible for lactic acidosis-induced vascular smooth muscle cell impairment in sepsis. Journal of Biomedical Science, 31, 3.

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Mitochondrial Respiration Profiling in Pancreatic Cancer

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Seahorse assay was performed to detect mitochondrial respiratory function. After transfection with siRNA, pancreatic cancer cells (PANC1 and BxPC3, 10000 cells per well) were seeded on seahorse cell culture plates (Agilent Technologies, Seahorse XF96 V3 PS Cell Culture Microplates). The XF Calibrant was prepared for calibration of the Seahorse XFe96 Analyser. On the next day, the cells were incubated for 1 h at 37 °C without CO₂. After calibrating the Seahorse XFe96 Analyser, the oxygen consumption rate (OCR) (performed by Agilent Technologies, Seahorse XF Cell Mito Stress Test Kit) was automatically analysed by a Seahorse XFe96 Analyser following the manufacturer’s instructions using oligomycin (1.5 μM), FCCP (2 μM), and rotenone (0.5 μM). The cells were then stained with Hoechst 33342 and counted by a Cytation™ 7 Cell Imaging Multi-Mode Reader. The OCR results were standardized by cell counts.

Qin C., Wang Y., Zhao B., Li Z., Li T., Yang X., Zhao Y, & Wang W. (2023). STOML2 restricts mitophagy and increases chemosensitivity in pancreatic cancer through stabilizing PARL-induced PINK1 degradation. Cell Death & Disease, 14(3), 191.

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Mitochondrial Function Evaluation

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OCR was determined using the Mito Stress Test Kit and XFe96 Extracellular Flux Analyzer (Seahorse Bioscience, Billerica, MA, USA). Cells were seeded in Seahorse XF96 V3 PS Cell Culture Microplates (Agilent Technologies, UK). Following incubation, cells were transferred to unbuffered XF assay media at pH 7.4 supplemented with 25 mM glucose and 1 mM sodium pyruvate and placed at 37 °C in a non-CO₂ incubator for one hour prior to assay. Cells were analysed using the Seahorse XF Cell Mito Stress Test (Agilent Technologies, UK). Immediately following each run cells were lysed, and protein concentration determined by Sulforhodamine B assay for normalisation. OCR was automatically calculated and analysed by the Seahorse Wave software (Agilent Technologies, UK).

Bi O., Anene C.A., Nsengimana J., Shelton M., Roberts W., Newton-Bishop J, & Boyne J.R. (2021). SFPQ promotes an oncogenic transcriptomic state in melanoma. Oncogene, 40(33), 5192-5203.

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Metabolic Profiling of Activated Monocytes

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All patients and healthy volunteers signed onto approved National Institutes of Health protocols. The study was conducted in accordance with the Declaration of Helsinki. Peripheral blood was collected in sodium heparin tubes. Peripheral blood mononuclear cells were isolated by density gradient centrifugation using Lymphocyte Separation Medium (Corning, catalog #MT25072CI). CD14⁺ selection was performed using magnetic beads (Miltenyi Biotec CD14 MicroBeads, catalog #130-050-201) following the manufacturer’s protocol. CD14⁺ monocytes were plated at 10⁵/50 μL per well in Seahorse XF96 V3 PS cell culture microplates (Agilent, 101085-004) in serum-free media. After 3 hours, 50 μL of complete media with 20% (2×) fetal bovine serum was added, and cells were rested overnight. Cells were then stimulated with media alone, IFNγ (1000 U/mL; Actimmune, NDC number 75987-111-10), and/or select inhibitors: rotenone (Sigma, #R8875), FK866 (Selleckchem, #S2799), diphenyleneiodonium (DPI) (Sigma, #D2926), rotenone and Antimycin A, oligomycin, or trifluoromethoxy carbonylcyanide phenylhydrazone (FCCP) (Agilent Seahorse Mito Stress Test, #103015-100).

McCann K.J., Christensen S.M., Colby D.H., McGuire P.J., Myles I.A., Zerbe C.S., Dalgard C.L., Sukumar G., Leonard W.J., McCormick B.A, & Holland S.M. (2022). IFNγ regulates NAD+ metabolism to promote the respiratory burst in human monocytes. Blood Advances, 6(12), 3821-3834.

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Mitochondrial Respiration Profiling

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Oxygen Consumption Rate (OCR) was determined using the Mito Stress Test Kit and XFe96 Extracellular Flux Analyzer (Seahorse Bioscience, Billerica, MA, USA). Cells were seeded in Seahorse XF96 V3 PS Cell Culture Microplates (Agilent Technologies, UK). Following incubation, cells were transferred to unbuffered XF assay media at pH 7.4 supplemented with 25 mM glucose and 1 mM sodium pyruvate and placed at 37°C in a non-CO₂ incubator for one hour prior to assay. Cells were analysed using the Seahorse XF Cell Mito Stress Test (Agilent Technologies, UK). Immediately following each run cells were lysed, and protein concentration determined by Sulforhodamine B assay for normalization. OCR was automatically calculated and analysed by the Seahorse Wave software (Agilent Technologies, UK).

Bi O., Anene C.A., Nsengimana J., Shelton M., Roberts W., Newton-Bishop J, & Boyne J.R. (2021). SFPQ promotes an oncogenic transcriptomic state in melanoma. Oncogene, 40(33), 5192-5203.

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Mitochondrial Respiration Profiling of Tumor Cells

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Cells were seeded and treated in 6-well plates as indicated, then lifted with trypsin and re-seeded onto Seahorse XF96 V3 PS Cell Culture Microplates (Agilent: 101085–004) overnight at experimentally optimized density. Extracellular flux analysis of oxygen consumption rate (OCR) was performed on the Seahorse Biosciences XF96 Flux Analyzer (Agilent) at baseline and after serial injection of oligomycin (1.5uM), FCCP (0.5 or 1uM), and antimycin A/rotenone (0.5uM) (Seahorse XF Cell Mito Stress Test Kit, Agilent: 103015–100) according to manufacturer’s recommendations. After analysis, tumor cell sample luciferase activity was determined for normalization using a SpectraMax i3 plate reader (Molecular Devices, Sunnyvale, CA). Data normalization, analysis, and calculation of maximum OCR were performed using Wave Desktop v2.6 (Agilent, RRID:SCR_014526).

Fox G.C., Su X., Davis J.L., Xu Y., Kwakwa K.A., Ross M.H., Fontana F., Xiang J., Esser A.K., Cordell E., Pagliai K., Dang H.X., Sivapackiam J., Stewart S.A., Maher C.A., Bakewell S.J., Sharma V., Achilefu S., Veis D.J., Lanza G.M, & Weilbaecher K.N. (2021). Targeted therapy to β3 integrin reduces chemoresistance in breast cancer bone metastases. Molecular cancer therapeutics, 20(6), 1183-1198.

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