Xf cell culture microplate

Manufactured by Agilent Technologies

Sourced in United States

The XF cell culture microplates are a product offered by Agilent Technologies for cell-based assays. The microplates are designed to facilitate the cultivation and analysis of cells in a controlled in vitro environment. The core function of these microplates is to provide a standardized and consistent platform for performing cell-based experiments and measurements.

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50 protocols using xf cell culture microplate

Metabolic Profiling of Expanded NK Cells

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For real-time analysis of the extracellular acidification rate (ECAR) and oxygen consumption rate (OCR) of purified and expanded NK cells cultured under various conditions, a Seahorse XF-24 Analyser, a Seahorse XFe-96 Analyser or a Seahorse XF-8 Analyser (Seahorse Bioscience) was used. In brief, 500,000 to 750,000 MACS purified, expanded NK cells were added to a 24-well XF Cell Culture Microplate, 100,000 to 200,000 MACS purified NK cells to a 96-well XFe Cell Culture Microplate and 200,000 ex vivo pure NK cells to an 8-well XF Cell Culture Microplate (Seahorse Biosciences). All cell culture plates were treated with Cell-Tak™ (BD Pharmingen) to ensure that the NK cells adhere to the plate. Sequential measurements of ECAR and OCR following addition of the inhibitors (Sigma) oligomycin (2 μM), FCCP (1 μM), rotenone (100 nM) plus antimycin A (4 μM), and 2-deoxyglucose (2DG, 30 mM) allowed for the calculation of basal glycolysis, glycolytic capacity, basal mitochondrial respiration, and maximal mitochondrial respiration. Where indicated, BMS303141 (10 μM, Sigma), SB204990 (30 μM, Tocris), BPTES (10 μM,Tocris) or an equivalent amount of vehicle control was injected into the Seahorse plate.

Loftus R.M., Assmann N., Kedia-Mehta N., O’Brien K.L., Garcia A., Gillespie C., Hukelmann J.L., Oefner P.J., Lamond A.I., Gardiner C.M., Dettmer K., Cantrell D.A., Sinclair L.V, & Finlay D.K. (2018). Amino acid-dependent cMyc expression is essential for NK cell metabolic and functional responses in mice. Nature Communications, 9, 2341.

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Adipocyte Respiration in Inguinal WAT

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Primary SVF cells from inguinal WAT were isolated and cultured for 3 days before plated in XF cell culture microplates (Seahorse Bioscience). SVF cells (10,000 cells) were seeded in each well and each sample has 8 replicates. After 6 days of differentiation, cultured adipocytes were washed twice and pre-incubated in XF medium for 1–2 h at room temperature. The oxygen consumption rate was measured by the XF extracellular flux analyzer (Seahorse Biosciences). The chemicals (final concentration, 0.2 mM Palmitate: 34 μM BSA, 2 μM Rotenone) were preloaded into cartridges and injected into XF wells in succession. OCR was calculated as a function of time (picomoles per minute).

Bi P., Shan T., Liu W., Yue F., Yang X., Liang X.R., Wang J., Li J., Carlesso N., Liu X, & Kuang S. (2014). Notch signaling regulates adipose browning and energy metabolism. Nature medicine, 20(8), 911-918.

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Cellular Assays for Neuroprotection

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6-OHDA, dimethyl sulfoxide (DMSO), paraformaldehyde (PFA), and thiazolyl blue tetrazolium bromide (MTT) were purchased from Sigma-Aldrich (St. Louis, MO, USA). A lactate dehydrogenase (LDH) kit and cocktail were purchased from Roche Applied Science (Indianapolis, IN, USA). F-12K medium, FBS, HS, penicillin-streptomycin (PS), trypsin-EDTA, and PBS were purchased from Life Technologies (Grand Island, NY, USA). Enhanced chemiluminescence (ECL) solution was obtained from Thermo Fisher Scientific (Rockford, IL, USA). RIPA lysis buffer was bought from Beyotime Biotechnology (Shanghai, China). H-89 was purchased from Selleck Chemicals (Shanghai, China). SYBR® Premix Ex Taq™ II kit was purchased from TaKaRa. (Dalian, China). Antibodies against p-PKA, PKA, p-Akt, Akt, p-CREB, CREB, p-PI3K, PI3K, p-GSK-3β, GSK-3β, β-actin, lamin B1, and HRP-conjugated anti-rabbit IgG were purchased from Cell Signaling Technology (Boston, MA, USA). Antibodies against Nrf2, PGC-1α, NRF1, and TFAM were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). XF cell culture microplates and XF96 extracellular flux assay kits were purchased from Seahorse Bioscience (North Billerica, MA, USA). All other chemicals of analytical grade were purchased from local sources.

Zhou H., Shao M., Yang X., Li C., Cui G., Gao C., Di L., Zhong H., Wang Y., Zhang Z, & Lee S.M. (2019). Tetramethylpyrazine Analogue T-006 Exerts Neuroprotective Effects against 6-Hydroxydopamine-Induced Parkinson's Disease In Vitro and In Vivo. Oxidative Medicine and Cellular Longevity, 2019, 8169125.

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Mitochondrial Respiration in Adipocytes

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Primary SVF cells from BAT and sWAT were isolated and cultured for 3 days before being plated in XF cell culture microplates (Seahorse Bioscience). SVF cells (10,000 cells) were seeded in each well, and each treatment included cells from three BAT or sWAT replicates. After 6-day differentiation, cultured adipocytes were washed twice and pre-incubated in XF medium (supplemented with 25 mM glucose, 2 mM glutamine and 1 mM pyruvate) for 1–2 h at 37 °C without CO₂. The OCR was measured using the XF Extracellular Flux Analyser (Seahorse Biosciences). Oligomycin (2 mM), FCCP (2 mM), and Antimycin A (0.5 mM) were preloaded into cartridges and injected into XF wells in succession. OCR was calculated as a function of time (pmoles per minute per μg protein).

Liu P., Huang S., Ling S., Xu S., Wang F., Zhang W., Zhou R., He L., Xia X., Yao Z., Fan Y., Wang N., Hu C., Zhao X., Tucker H.O., Wang J, & Guo X. (2019). Foxp1 controls brown/beige adipocyte differentiation and thermogenesis through regulating β3-AR desensitization. Nature Communications, 10, 5070.

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Metabolic Profiling of Immune Cells

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Cells were plated into XF Cell Culture Microplates (Seahorse Bioscience) overnight at 37°C and 5% CO₂. The next day, cells were treated with IL4 and/or IL13 for 1 h. To measure OCR and ECAR, media were replaced in the Seahorse microplates with assay medium free of sodium bicarbonate and FBS, and the plate was incubated in a CO₂-free incubator for 1 h at 37°C. Oligomycin, FCCP, and rotenone were sequentially injected at a final concentration of 2 μg/ml, 1 μM and 1 μM, respectively. Experiments were run using an XF analyzer, and raw data were normalized with total protein measured in each well of the microplate.

Dey P., Li J., Zhang J., Chaurasiya S., Strom A., Wang H., Liao W.T., Cavallaro F., Denz P., Bernard V., Yen E.Y., Genovese G., Gulhati P., Liu J., Chakravarti D., Deng P., Zhang T., Carbone F., Chang Q., Ying H., Shang X., Spring D.J., Ghosh B., Putluri N., Maitra A., Wang Y.A, & DePinho R.A. (2020). Oncogenic Kras driven metabolic reprogramming in pancreas cancer cells utilizes cytokines from the tumor microenvironment. Cancer discovery, 10(4), 608-625.

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Adipocyte Respiration in Inguinal WAT

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Measuring Adipocyte Respiration via Seahorse

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Primary brown preadipocytes were seeded (10,000 cells/well) and differentiated in XF cell culture microplates (Seahorse Bioscience, Billerica, MA, USA) as described above. Cells were transfected with miR-26a mimics or NCs for 48 h before performing the assay. Adipocytes were washed twice and maintained in XF assay medium (25 mM glucose, 2 mM glutamine, 1 mM sodium pyruvate) for 1 h at 37°C without CO₂. OCR was measured by XF Extracellular Flux Analyzer (Seahorse Biosciences). During measurement, cells were treated with oligomycin (2.5 μM), carbonyl cyanide 4-(trifluoromethoxy) phenylhydrazone (FCCP, 2 μM), and antimycin A (0.5 μM) and rotenone (0.5 μM) in succession.

Xu H., Du X., Xu J., Zhang Y., Tian Y., Liu G., Wang X., Ma M., Du W., Liu Y., Dai L., Huang W., Tong N., Wei Y, & Fu X. (2020). Pancreatic β cell microRNA-26a alleviates type 2 diabetes by improving peripheral insulin sensitivity and preserving β cell function. PLoS Biology, 18(2), e3000603.

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Oxygen Consumption Profiling of Mouse SAT-derived SVF Cells

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Mouse primary SAT-derived SVF was isolated from WT and UCP1 KO mice and seeded into 0.2% gelatin (wt/vol) coated 24-well XF cell culture microplates (Seahorse Bioscience, Billerica, MA, USA). Differentiated SVF cells were treated with or without 100 μM EPA. Then, culture media were changed to the XF assay media (Seahorse Bioscience, Billerica, MA, USA) containing 2 mmol/L sodium pyruvate and 25 mmol/L glucose and placed in a 37 °C non-CO₂ incubator for 1 h. To determine oxygen consumption rate (ORC) changes in the differentiated SVF cells, an XF Cell Mito Stress Test Kit (Agilent, Santa Clara, CA, USA) was used and oligomycin A (1 mmol/L), carbonyl cyanide-4-(trifluoromethoxy) phenylhydrazone (FCCP, 0.3 mM), and antimycin/rotenone (A/R, 1 mM each) were injected according to the manufacturer’s instructions. Respiration profiles were calculated by the following formula:

Zu Y., Pahlavani M., Ramalingam L., Jayarathne S., Andrade J., Scoggin S., Festuccia W.T., Kalupahana N.S, & Moustaid-Moussa N. (2023). Temperature-Dependent Effects of Eicosapentaenoic Acid (EPA) on Browning of Subcutaneous Adipose Tissue in UCP1 Knockout Male Mice. International Journal of Molecular Sciences, 24(10), 8708.

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Osteoclast Metabolic Profiling by Seahorse Assay

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Osteoclast precursors were seeded at a density of 1 × 10⁵ cells/well with α-MEM supplemented with M-CSF (30 ng/ml) in XF cell culture microplates (Seahorse Bioscience, Billerica, MA, USA) and allowed to attach overnight. The next day, cells were incubated in sodium bicarbonate-free α-MEM media (Hyclone, Logan, UT, USA) buffered with 10 mM HEPES (adjusted to pH 7.4 with 1 M NaOH) [18 (link)] and supplemented with 10% FBS and antibiotics in the presence of M-CSF (30 ng/ml) alone or both M-CSF (30 ng/ml) and RANKL (100 ng/ml) for 1 h. OCR and ECAR were measured continuously at 37°C using an XF96 analyzer (Seahorse Bioscience), and the readings were collected every 8 min after mixing, waiting, and recording periods in each well. Values obtained were the average of readings for 3 h.

Ahn H., Lee K., Kim J.M., Kwon S.H., Lee S.H., Lee S.Y, & Jeong D. (2016). Accelerated Lactate Dehydrogenase Activity Potentiates Osteoclastogenesis via NFATc1 Signaling. PLoS ONE, 11(4), e0153886.

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Seahorse XF24 Assay for Cellular Respiration

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Cell monolayers were cultured in XF Cell Culture Microplates (Seahorse Bioscience, North Billerica, MA, USA) at a density of 2.5 × 10⁴ cells per well. The sensor cartridge (Seahorse Bioscience) was polarized overnight and calibrated. After ZVI-NP treatment, the medium was replaced with appropriate assay medium without sodium bicarbonate and serum, and cells were then incubated for 30 min at 37 °C without CO₂. The compounds were injected sequentially: 1 μM oligomycin; 2 μM FCCP; 2 μM Rotenone (all from Sigma-Aldrich). The basal OCR and OCR responses toward compounds injection were performed in a Seahorse XF24 analyzer (Seahorse Bioscience) according to the manufacturer's instructions.

Hsieh C.H., Hsieh H.C., Shih F.H., Wang P.W., Yang L.X., Shieh D.B, & Wang Y.C. (2021). An innovative NRF2 nano-modulator induces lung cancer ferroptosis and elicits an immunostimulatory tumor microenvironment. Theranostics, 11(14), 7072-7091.

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