Xf96 cell culture microplate

Manufactured by Agilent Technologies

Sourced in United States

The XF96 cell culture microplates are a product offered by Agilent Technologies. They are designed for use in cell-based assays and provide a standardized platform for measuring cellular parameters such as oxygen consumption rate and extracellular acidification rate.

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Seahorse XF96 cell culture microplates (87 citations) XF96 microplates (46 citations) XF96 polystyrene cell culture microplates (13 citations) Cell culture microplate (12 citations) XF96 V3 PS cell culture microplates (11 citations) Seahorse XF96 V3 PS Cell Culture Microplates (6 citations) Cell-Tak™ coated XF96 polystyrene cell culture microplates (4 citations) Agilent Seahorse XF96 Cell Culture Microplate (4 citations) XF96-well cell culture microplates (3 citations) XF96 culture microplates (2 citations)

132 protocols using xf96 cell culture microplate

Mitochondrial Respiration Profile Measurement

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OCR was assessed in real-time with a Seahorse XF96 Extracellular Flux Analyzer (Seahorse Bioscience, Billerica, MA, USA), which allows to measure OCR changes after sequential addition of modulators of respiration that target components of the electron transport chain in mitochondria. Cells (1 × 10⁴ cells/well/200 µL of DMEM) were plated in a XF 96 cell culture microplate (Seahorse Bioscience Inc., Billerica, MA, USA). Cells were washed with base media once, immersed in 175 µL base media, and incubated in the absence of CO₂ for 20 min. After baseline measurements, respiration was measured after sequentially adding 25 µL of oligomycin (inhibitor of ATP synthase, 1 µg/mL), carbonyl cyanide-4 (trifluoromethoxy) phenylhydrazone (FCCP) (a protonophore and uncoupler of mitochondrial oxidative phosphorylation, 0.5 μM), and a combination of rotenone (mitochondrial complex I inhibitor, 1 μM) and antimycin A (mitochondrial complex III inhibitor, 1 μM) for OCR measurement using the XF Cell Mito Stress Test Kit (Cat. No. 103015-100, Seahorse Bioscience Inc., Billerica, MA, USA). OCR values were normalized for the protein content of each sample and expressed as the unit of pmoles/min. Basal OCR was expressed as percentage of the untreated cells (None). Basal OCR for “None” was set at 100.

Jeong Y., Lim J.W, & Kim H. (2019). Lycopene Inhibits Reactive Oxygen Species-Mediated NF-κB Signaling and Induces Apoptosis in Pancreatic Cancer Cells. Nutrients, 11(4), 762.

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Evaluating Cellular Metabolism using Seahorse Assay

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hES cells were cultured on feeder cells in XF96 cell culture microplate (Seahorse Bioscience). HEK293 and Hela cells were seeded onto a Matrigel-coated XF96 cell culture microplate. ECAR and OCR were measured using a standard protocol (Seahorse Bioscience) and were normalized using counted cell numbers.

Fang Y., Liu Z., Chen Z., Xu X., Xiao M., Yu Y., Zhang Y., Zhang X., Du Y., Jiang C., Zhao Y., Wang Y., Fan B., Terheyden-Keighley D., Liu Y., Shi L., Hui Y., Zhang X., Zhang B., Feng H., Ma L., Zhang Q., Jin G., Yang Y., Xiang B., Liu L, & Zhang X. (2017). Smad5 acts as an intracellular pH messenger and maintains bioenergetic homeostasis. Cell Research, 27(9), 1083-1099.

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Mitochondrial Respiration and Glycolysis Profiling

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OCR, a measure of mitochondrial respiration, and ECAR, a measure of glycolysis, were determined using an XF96 extracellular flux analyzer (Seahorse Bioscience). Transfected 3T3-L1 cells were seeded in a 96-well XF96 cell culture microplate (Seahorse Bioscience) at a density of 7,000 cells per well in 200 μl of DMEM (4.5 g/l glucose) supplemented with 10% FBS (Sigma) 36 h before measurement. Six replicates per cell type were included in the experiments, and four wells were chosen evenly in the plate to correct for temperature variation. On the day of the assay, the growth medium was exchanged for assay medium (unbuffered DMEM with 4.5 g/l glucose). Oligomycin at a final concentration of 2.0 μM, FCCP (carbonyl cyanide-p-trifluoromethoxyphenylhydrazone) at 1.0 μM, 2-deoxyglucose at 100 mM, and rotenone at 15.0 μM were sequentially injected into each well in accordance with the manufacturer’s protocol. Basal mitochondrial respiration, maximal respiration, ATP production, and basal glycolysis were determined according to the manufacturer’s instructions. At the conclusion of the assay, cells in the analysis plate were lysed using CelLytic M (Sigma). Protein concentration was measured using the Bradford assay^{59 (link)} and used to normalize the bioenergetic profile data.

Minster R.L., Hawley N.L., Su C.T., Sun G., Kershaw E.E., Cheng H., Buhule O.D., Lin J., Reupena M.S., Viali S., Tuitele J., Naseri T., Urban Z., Deka R., Weeks D.E, & McGarvey S.T. (2016). A thrifty variant in CREBRF strongly influences body mass index in Samoans. Nature genetics, 48(9), 1049-1054.

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Measuring Oxygen Consumption Rates in Cells

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The oxygen consumption rates (OCR) of primary mouse AECIIs, SAECs, and
MLE-12 cells were measured by using a Seahorse XF96 Extracellular Flux Analyzer
(Seahorse Bioscience, Billerica, MA, USA), as previously described^{62 (link)}. All assays were performed
using a seeding density of 60,000 cells/well in 200μl of DMEM in a XF96
cell culture microplate (Seahorse Bioscience). After the cells were switched to
unbuffered DMEM supplemented with 2 mM sodium pyruvate and 20mM carnosine 1h
prior to the beginning of the assay and maintained at 37 °C. OCR was
measured after sequentially adding to each well 25μl of oligomycin (an
ATP-synthase inhibitor), FCCP (a protonophore) and rotenone (inhibitors of
complex I and III), to reach working concentrations of 1μg/ml,
1μM and 0.5μM respectively. OCR is reported in
picomoles/minute/60,000 cells.

Yu G., Tzouvelekis A., Wang R., Herazo-Maya J.D., Ibarra G.H., Srivastava A., de Castro J.P., DeIuliis G., Ahangari F., Woolard T., Aurelien N., e Drigo R.A., Gan Y., Graham M., Liu X., Homer R.J., Scanlan T.S., Mannam P., Lee P.J., Herzog E.L., Bianco A.C, & Kaminski N. (2017). Thyroid hormone inhibits lung fibrosis in mice by improving epithelial mitochondrial function. Nature medicine, 24(1), 39-49.

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Glycolytic Capacity Measurement in Cells

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The shPA200 or control cells were seeded (3.5 × 10⁴ cell/well) into an XF-96 cell-culture microplate (Seahorse Bioscience, Agilent, Chicopee, MA, USA). Cells were incubated overnight at 37 °C in a 5% CO₂ incubator. The next day, the growth medium was replaced by 180 µL XF glucose-free assay medium (Seahorse Bioscience, Agilent, Chicopee, MA, USA) and incubated at 37 °C in a CO₂-free, humidified incubator for 60 min. In parallel, the pre-incubated (overnight) XF-96 sensor cartridge was loaded for calibration (20 min). The extracellular acidification rate (ECAR) baseline was determined five times (5 min each), and then the drugs were sequentially injected at the following final concentrations: 10 mM glucose (Glu), 1 µM oligomycin (Olig), and 50 mM 2-deoxy-glucose (2-DG). The measurement of ECAR took place five times (5 min each) in each phase of injection. The ECAR data were normalized to total protein amount in each well. The protein concentration was measured as described above. Data analysis was performed using Wave 2.3 Agilent Seahorse desktop software. Statistical analyses were assessed using Graphpad Prism 8.2.1 software.

Douida A., Batista F., Boto P., Regdon Z., Robaszkiewicz A, & Tar K. (2021). Cells Lacking PA200 Adapt to Mitochondrial Dysfunction by Enhancing Glycolysis via Distinct Opa1 Processing. International Journal of Molecular Sciences, 22(4), 1629.

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Analyzing Mitochondrial Function in SK-N-SH Cells

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SK-N-SH cells transfected with SSOs for 24 h were trypsinized and seeded in a Seahorse XF96 cell culture microplate (2 × 10⁴ cells/well). After incubation for 24 h, the medium was replaced with Seahorse XF medium and incubated for 1 h at 37 °C in a non-CO₂ incubator. The oxygen consumption rate (OCR) was measured using a Seahorse XFe96 Analyzer (Agilent, CA, USA) with the Mito Stress Test Kit according to the manufacturer's instructions. The measurements were normalized to the cell numbers.

Cho N., Joo J., Choi S., Kang B.G., Lee A.J., Youn S.Y., Park S.H., Kim E.M., Masliah E., Ko Y., Cha S.S., Jung I, & Kim K.K. (2023). A novel splicing variant of DJ-1 in Parkinson's disease induces mitochondrial dysfunction. Heliyon, 9(3), e14039.

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Metabolic Profiling of OS Cells

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The real-time glycolytic rate (ECAR) and oxygen consumption rate (OCR) of OS cells were analyzed by an XF96 metabolic flux analyzer (Seahorse Biosciences, Billerica, MA, USA) as previously described [19 (link)]. Briefly, 3 × 10⁴ target cells were seeded into each well of a Seahorse XF 96 cell culture microplate. After the probes were calibrated, for OCR, 1 mM oligomycin, 1 mM p-trifluoromethoxy carbonyl cyanide phenylhydrazone (FCCP) and 2 mM antimycin A plus 2 mM rotenone (Rote/AA) were sequentially injected; and for ECAR, 10 mM glucose, 1 mM oligomycin, and 80 mM 2-deoxyglucose (2-DG) were sequentially injected. Data were assessed and analyzed by using Seahorse XF-96 Wave software.

Shen Y., Zhao S., Wang S., Pan X., Zhang Y., Xu J., Jiang Y., Li H., Zhang Q., Gao J., Yang Q., Zhou Y., Jiang S., Yang H., Zhang Z., Zhang R., Li J, & Zhou D. (2018). S1P/S1PR3 axis promotes aerobic glycolysis by YAP/c-MYC/PGAM1 axis in osteosarcoma. EBioMedicine, 40, 210-223.

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Metabolic Profiling of B Cell Subsets

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Flow-sorted B cell populations (resting B cells, activated B cells and
plasmablasts) from at least 3 Py-infected (day 10 p.i.) were
plated at 250,000 cells per well in poly-lysine coated Seahorse XF96 cell
culture microplate. Cells were allowed to adhere for 30 min to 1 h. OCR was
measured in modified DMEM containing 2 mM L-glutamine (XF media) under basal
conditions in a 96-well extracellular flux assay using a Xfe-96 (Seahorse
Bioscience). In experiments comparing metabolic activity of B cell populations
sorted from mice maintained on either regular water or water supplemented with
L-glutamine, the culture medium was devoid of L-glutamine.

Vijay R., Guthmiller J.J., Sturtz A.J., Surette F.A., Rogers K.J., Sompallae R.R., Li F., Pope R.L., Chan J.A., de Labastida Rivera F., Andrew D., Webb L., Maury W.J., Xue H.H., Engwerda C.R., McCarthy J.S., Boyle M.J, & Butler N.S. (2020). Infection-induced plasmablasts are a nutrient sink that impairs humoral immunity to malaria. Nature immunology, 21(7), 790-801.

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Measuring Cellular Bioenergetics in vitro

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Cells were plated at 80% confluence in a Seahorse XF96 cell culture microplate. After 16 hours, medium was replaced with medium containing DMSO or ESI-09. After 24 hours, medium was replaced with Seahorse XF assay medium pH 7.4 (unbuffered DMEM containing 200 mM GlutaMax-I and glucose) and the plate was incubated at 37°C for 1 hour. Baseline and maximal oxygen consumption rates and extracellular acidification rates were measured using XFp Extracellular Flux Analyzer (Agilent, Santa Clara, CA). The stressor mix contained FCCP (0.5 μM), an oxidative phosphorylation uncoupler and oligomycin (1 μM), an ATP synthase inhibitor.

Krishnan A., Bhasker A.I., Singh M.K., Rodriguez C.I., Castro-Pérez E., Altameemi S., Lares M., Khan H., Ndiaye M., Ahmad N., Schieke S.M, & Setaluri V. (2022). EPAC Regulates Melanoma Growth by Stimulating mTORC1 Signaling and Loss of EPAC Signaling Dependence Correlates with Melanoma Progression. Molecular cancer research : MCR, 20(10), 1548-1560.

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Macrophage Glycolysis and Mitochondrial Respiration

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Macrophages were plated in the specialized XF96 cell culture microplate (Seahorse Bioscience) at 8 × 10⁴ cells/well. Cells were exposed to LPS (100 ng/mL, 24h) before glycolysis stress assay to measure extracellular acidification rate (ECAR) and mito-stress assay for oxygen consumption rate (OCR) by Seahorse Bioscience Extracellular Flux Analyzer (Agilent) as described (Chen et al., 2019 (link)). The cells were then lysed in RIPA buffer and subjected to Lowry protein assay (Bio-Rad). ECAR and OCR values were normalized to protein content.

Zhang J., Chang J., Beg M.A., Huang W., Zhao Y., Dai W., Wu X., Cui W., Pillai S.S., Lakhani H.V., Sodhi K., Shapiro J.I., Sahoo D., Zheng Z., Silverstein R.L, & Chen Y. (2022). Na/K-ATPase suppresses LPS-induced pro-inflammatory signaling through Lyn. iScience, 25(9), 104963.

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