OCR and ECAR measurements were performed by using the Seahorse XF24 Extracellular Flux Analyzer (Seahorse, USA). After trypsinization, cancer cells were seeded in Seahorse XF24 cell plates (2.5 × 104/well) in a humidified 37 °C, 5% CO2 incubator for 24–48 h. The cell plates were then placed in a 37 °C, 0% CO2 incubator for 60 min prior to the start of an assay. A glycolytic stress test kit (Seahorse) and mitochondrial stress test kit (Seahorse) were used to detect glycolytic flux and mitochondrial metabolic flux, respectively, following the manufacturer’s protocols.
Xf24 cell plates
Manufactured by Agilent Technologies
The XF24 cell plates are a type of multi-well plate designed for use with Agilent's XF Extracellular Flux Analyzers. The plates provide a standardized platform for culturing and analyzing cells in a high-throughput manner.
Automatically generated - may contain errors
Lab products found in correlation
Xf24 extracellular flux analyzer, by Agilent Technologies (2 mentions)
Glycolytic stress test kit, by Agilent Technologies (1 mentions)
Mitochondrial stress test kit, by Agilent Technologies (1 mentions)
Glycolysis stress test kit, by Agilent Technologies (1 mentions)
Seahorse base medium, by Agilent Technologies (1 mentions)
Xf24 analyser, by Agilent Technologies (1 mentions)
Oligomycin a, by Merck Group (1 mentions)
Antimycin a, by Merck Group (1 mentions)
Rotenone, by Merck Group (1 mentions)
Seahorse xf24 analyzer, by Agilent Technologies (1 mentions)
5 protocols using xf24 cell plates
1
Cancer Cell Metabolic Profiling
2
Glucose Flux Assay in Transfected HeLa Cells
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XF glucose flux assays were performed using an XF24 Extracellular Flux Analyzer (Seahorse Bioscience, North Billerica, MA, USA). That is based upon fluorimetric detection of O2 and H+ levels via solid-state probes on a sensor cartridge53 (link). HeLa cells, seeded at 10,000 cells/well in XF24 cell plates (Seahorse Bioscience), were transfected with expression vectors for GKRP (WT), GKRP (K5R), GKRP (K5Q), GK, p300, SIRT2 (WT) and/or SIRT2 (H187Y). The following day, the media was changed to DMEM (without serum, glucose or bicarbonate, but with 2 mM glutamine), and incubated 1 h before the assay in a non-CO2 incubator at 37 °C. Injections of glucose (10 mM final), oligomycin (2.5 μM final) and 2-deoxyglycose (0.1 M final) were then diluted in DMEM media and loaded into ports A, B, and C sequentially. Reagents were optimized using a Glycolysis Stress Test kit (Seahorse Bioscience), using the XF24 analyzer protocol and algorithm. Each assay was run in one plate with 3–4 replicates, and repeated at least 5 times.
3
Metabolic Profiling of Oligodendrocytes
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Following papain dissociation, cells were plated onto XF24 cell plates (Seahorse Bioscience, Billerica, MA) pre-coated with matrigel, laminin and fibronectin (as above). Optimization of reagents was performed and the protocols and algorithm program were used according to the XF24 analyser. Briefly, cells were transitioned from their oligodendrocyte specific media into the Agilent Seahorse Base Medium which has low buffering capacity and 0 mM glucose and maintained in ambient CO2 for 25 min. The plate was then inserted into the machine; baseline oxygen consumption rate and extracellular acidification rate were measured prior to the addition of glucose (10 mM), oligomycin (1.5 μM) and 2-deoxy-glucose (100 mM). Both oxygen consumption rate and extracellular acidification rate were measured three times following the injection of each drug.
4
Metabolic Profiling of Macrophages
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The metabolic measurements were performed as previously described (39 (link)). Briefly, 14 day old MDM were re-seeded in XF24 cell plates (Agilent Technologies) at 150,000 MDMs/well. Cultures were then challenged with bacteria or mock-infected were washed twice with XF medium supplemented with 4.5 g/L D-glucose, 2.0 mM L-glutamine, 1.0 mM sodium-pyruvate, 100 U/L penicillin and 100 μg/mL streptomycin at pH 7.4 (adjusted with 1.0 M NaOH). Next 630 μL modified XF medium was added to each well and incubated for 1 h at 37·C without CO2. 70 μL oligomycin A (15 μM), 77 μL FCCP (20 μM) and 85 μL rotenone (10 μM) plus antimycin A (10 μM) (Sigma-Aldrich) were added to the cartridge injection ports A, B and C, respectively and incubated for 1 h at 37°C without CO2. The plate was loaded into the XF24 analyser (Seahorse, Agilent Technologies). After equilibration, the cartridge containing the oxygen sensor, measuring the oxygen consumption rate (OCR) and the cartridge containing the proton sensor, measuring the extracellular acidification rate (ECAR) kinetics were run before and after injecting oligomycin, FCCP and rotenone plus antimycin A, respectively. Data were normalized by total protein.
5
Mitochondrial Respiration Assay Protocol
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One day before the experiment, on four different days, 60,000 cells were plated on XF24 cell plates (Agilent) to measure cell respiration. After irradiation, PBS was replaced by DMEM without sodium bicarbonate, and cells were incubated for 1 h at 37°C and atmospheric pressure of CO2. Oxygen consumption rate (OCR) was measured in a Seahorse XF24 Analyzer (Agilent), before and after subsequent additions of 1 μM oligomycin, 1 μM CCCP and a mix of 1 μM antimycin and 1 μM rotenone. Each compound was added after three cycles of measurements of 3 min each. The concentration of CCCP was determined through the previous titration. At the end of the experiments, each well was washed once with PBS and proteins were resuspended in 100 μM ammonium bicarbonate, containing 8 M urea and 1% sodium deoxycholate. After homogenization, protein concentration was determined by using a BCA assay kit. The OCR values were normalized by the amount of total protein in each well. The number of biological replicates is described in the legend of Figure 8 .
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