Cells were harvested and resuspended at 5x106 cells/mL in appropriate Seahorse media. 40μL of cells were added to 140μL of appropriate seahorse media and plated in XF96 cell culture microplates that were precoated with 25μL of Cell tak reagent overnight according to manufacturer’s instructions (Corning Inc., Corning, NY, #354240). For examining mitochondrial function, a mito-stress kit was used with additions of the following reagents to cells in appropriate Seahorse media: 1 μM Oligomycin, 1μM fluoro-carbonyl cyanide phenylhydrazone (FCCP, an uncoupler of respiration and oxidative ATP synthesis), and pre-mixed 0.5 μM rotenone + antimycin A solution (Electron Transport Chain complex I and III inhibitors). Maximum respiratory capacity was calculated as the difference between the oxygen consumption rate (OCR) after addition of FCCP and the OCR after addition of rotenone/antimycin A. Spare respiratory capacity was calculated as the difference between maximal (post FCCP) and basal respiration. Plates were run on a Seahorse XF96 Extracellular Flux Analyzer according to the manufacturer’s protocols.
Xf96 cell culture microplate
Manufactured by Corning
Sourced in United States
The XF96 cell culture microplates are a laboratory product designed for the culturing and analysis of cells. The plates feature a 96-well format and are suitable for a variety of cell-based assays and experiments.
Automatically generated - may contain errors
Lab products found in correlation
Xfe96 analyzer, by Agilent Technologies (4 mentions)
Cell tak cell and tissue adhesive, by Agilent Technologies (2 mentions)
Cell tak, by Corning (2 mentions)
Cell tak reagent, by Corning (1 mentions)
Xf96 extracellular flux analyzer, by Agilent Technologies (1 mentions)
Cell tak cell and tissue adhesive, by Corning (1 mentions)
Lsrfortessa, by BD (1 mentions)
Ab65331, by Abcam (1 mentions)
Cell tak solution, by Corning (1 mentions)
Fluoro carbonyl cyanide phenylhydrazone (fccp), by Merck Group (1 mentions)
Bone morphogenetic protein 4 (bmp4), by Thermo Fisher Scientific (1 mentions)
Seahorse xf cell mito stress test, by Agilent Technologies (1 mentions)
Xf cell mito stress test, by Agilent Technologies (1 mentions)
Xf rpmi medium, by Agilent Technologies (1 mentions)
Seahorse xf cell mito stress test kit, by Agilent Technologies (1 mentions)
8 protocols using xf96 cell culture microplate
1
Mitochondrial Function Analysis via Seahorse Assay
2
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.
3
Monocyte Glucose Uptake Dynamics
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Glucose uptake in monocytes was measured by flow cytometry. Cryopreserved MNCs were rested for 1 h at 37 °C in cRPMI after gentle thawing, and then counted before stimulation for 2 h and 40 min in presence of LPS (10 ng ml-1), with 2-(N-[7-Nitrobenz-2-oxa-1,3-diazol-4-yl]Amino)-2-Deoxyglucose (2-NBDG, 15 μM) added for the last 40 min. Cells were stained with CD14 (eBioscience #25-0149; 1:100 dilution) and CD16 (eBioscience #48-0168; 1:100 dilution) at the beginning of LPS stimulation. Cells were washed twice before acquisition on a LSRFortessa™ flow cytometer (Becton Dickinson). Data was analyzed using FlowJo® (FlowJo, LLC). For extracellular flux analyses, monocytes were plated on Cell-Tak™ (Corning, #354240)-coated XF96 Cell Culture Microplates. Cells were rested in a non-CO2 incubator, and glycolysis stress test was performed as per manufacturer protocol (Agilent Seahorse, #103020-100) using the Seahorse XFe96 Analyzer. Extracellular acidification rate (ECAR) was normalized to protein content. L-lactate was measured by colorimetry (Abcam, #ab65331).
4
Metabolic Profiling of Activated Human CD4 T Cells
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Human CD4 T cells were expanded in culture for 10–12 days and pre-treated overnight with various inhibitors. The next day, cells were counted via trypan blue and seeded at 125,000 cells/well in Seahorse XF96 Cell culture microplates, which were pre-treated with 22.4 μg/ml Cell-Tak solution for 20 min (Corning). Mito Stress tests were conducted according to the manufacturer's instructions (Agilent) with Seahorse Agilent pH7.4 media + 1 mM pyruvate + 10 mM glucose + 2 mM L-glutamine. Final concentrations of inhibitors after injections were 1.5 μM oligomycin A, 1.5 μM trifluoromethoxy carbonylcyanide phenylhydrazone (FCCP) (Sigma Aldrich), 0.5 μM rotenone and 0.5 μM antimycin A.
GAPDH activity was measured using the colorimetric Glyceraldehyde 2 Phosphate Dehydrogenase Activity Assay Kit (Abcam). 4 × 106 CD4 T cells were treated overnight with the various inhibitors or DMSO control, washed in cold PBS, and lysed in 150 μl of GAPDH buffer. Samples were measured in triplicate wells and the average value was plotted for each donor.
GAPDH activity was measured using the colorimetric Glyceraldehyde 2 Phosphate Dehydrogenase Activity Assay Kit (Abcam). 4 × 106 CD4 T cells were treated overnight with the various inhibitors or DMSO control, washed in cold PBS, and lysed in 150 μl of GAPDH buffer. Samples were measured in triplicate wells and the average value was plotted for each donor.
5
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.
6
Metabolic Profiling of CD4+ T Cells
7
Mitochondrial Respiration Profiling
8
Mitochondrial and Glycolytic Stress Assays
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Briefly, 3 × 104 Jurkat cells in 100 μl of medium were seeded in a Cell-Tak (Corning, NY, USA, 354240) coated XF96 Cell Culture microplate and treated with a final concentration of 10 μM Erlotinib and a corresponding amount of the DMSO control in culture media. After 24 hrs., cell culture media were replaced with Seahorse XF modified media. Cells were treated with 1 μM of oligomycin A, 0.6 μM FCCP, or mixture of 1 μM of rotenone and 1 μM of antimycin A in standard mitochondrial stress test conditions. For the glycolytic stress test, a similar plating procedure was followed and 10 mM glucose, 1 μM oligomycin, and 50 mM 2-Deoxy-D-glucose were added to wells.
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