Oxygen consumption rate (OCR) was analyzed in an X96 Extracellular Flux Analyzer with XF Cell Mito Stress Test Kit (Seahorse Biosciences) at 37°C. DA neurons were plated on Poly-D-Lysine coated XF96 cell culture plate at 40K per well and cultured for 7 days with 80ul neuronal culture medium/well. Four wells without neuron seeding from each plate were set as temperature and background control. For measurement, neurons were gently rinsed with 100ul/well assay medium (XF Base medium (Seahorse Biosciences) with 2mM Glutamine and 10mM glucose), put into 175ul/well fresh assay medium and assayed. Three baseline recordings were made, followed by sequential injection of the injection of the ATP synthase inhibitor oligomycin, the mitochondrial uncoupler Carbonyl cyanide-4-(trifluoromethoxy) phenylhydrazone (FCCP) and the Complex I inhibitor rotenone. Two minutes OCR measurement were performed at 3 minute intervals with mixing and each condition was measured in independent well.
X96 extracellular flux analyzer
Manufactured by Agilent Technologies
The X96 Extracellular Flux Analyzer is a lab equipment product designed for measuring extracellular metabolic parameters of cells in real-time. The device utilizes oxygen-sensing and pH-sensing technology to quantify oxygen consumption rate and extracellular acidification rate of cell samples.
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6 protocols using x96 extracellular flux analyzer
1
Mitochondrial Respiration in DA Neurons
2
Osteoblast Mitochondrial Respiration Analysis
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OCR was analyzed in an X96 Extracellular Flux Analyzer with XF Cell Mito Stress Test kit (Seahorse Biosciences) at 37°C. OBs isolated from OcnCre and Lrp4Ocn-cko mice were plated on XF96 cell culture plates at 40,000 per well and cultured for 3 d. As temperature and background controls, four wells without OB seeding from each plate were set. For measurement, OBs were gently rinsed with 100 µl/well XF Base medium with 2 mM glutamine and 10 mM glucose. 175 µl/well fresh assay medium was added and assayed. The ATP synthase inhibitor oligomycin, the mitochondrial uncoupler carbonyl FCCP, and the complex I inhibitor rotenone were sequentially injected, and three baseline recordings were made. OCR measurements were performed at 3-min intervals, and each condition was measured in an independent well.
3
Mitochondrial Respiration Profiling of Cells
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Mitochondrial function was evaluated by oxygen consumption rate (OCR). It was measured in an X96 Extracellular Flux Analyzer with XF Cell Mito Stress Test Kit (Seahorse Biosciences) at 37 °C. BMSCs and OBs were plated on XF96 cell culture plate at 10 K per well and cultured for overnight with 80ul culture medium/well. Four wells without cells seeding from each plate were set as temperature and background control. For measurement, cells were gently rinsed with 100ul/well assay medium (XF Base medium (Seahorse Biosciences) with 2 mM Glutamine and 10 mM glucose), then put into 175ul/well assay medium and assayed. Three baseline OCR were calculated, followed by sequential injection of the ATP synthase inhibitor oligomycin, the mitochondrial uncoupler Carbonyl cyanide-4- (trifluoromethoxy) phenylhydrazone (FCCP) and the Complex I inhibitor rotenone. Two minutes OCR measurement were made at 3 min intervals with mixing and each condition was measured in each well.
4
Bioenergetic Response to Drug Treatment
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The bioenergetic function of cells in response to drug treatments was determined using a Seahorse Bioscience X96 Extracellular Flux Analyzer (Seahorse Bioscience). 5,000 Cells were seeded in specialized V7 Seahorse tissue culture plates (Agilent, 102601 -100) and allowed to adhere overnight. In cases of ACY1215 pretreatment, cells were incubated in drug versus vehicle control for indicated for 24 hours prior to plating, and maintained in drug/vehicle during the plating process. One hour before the experiment, cells were washed with prewarmed RPMI-1640 and changed into fresh RPMI-1640 medium. Three baseline measurements were taken for oxygen consumption rate, followed by three measurements after injection of Rotenone (final concentration 20 uM) and Antimycin (20 uM). Following measurements, each cells from each well were counted for normalization. Basal oxygen consumption rate was measured by average of baseline measurements minus average of measurements following Rotenone/antimycin treatment.
5
Mitochondrial and Glycolytic Profiling of HK-2 Cells
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The seahorse Bioscience X96 extracellular flux analyzer (Agilent Technologies) was used to measure the rate change of dissolved O2 in the medium that immediately surrounded adherent cells cultured in the XF96 V3 cell culture microplate (Seahorse Bioscience). Then, HK-2 cells, which were cultured in RPMI 1640 supplemented with 0.5% FBS, were seeded in the XF96 V3 cell culture microplate at 0.8 × 104 cells per well. Afterwards, these cells were washed and incubated in the base medium (Agilent Technologies) at 37 °C for one hour. The oxygen consumption rates (OCR, pmol.min-1) was measured in real-time using a mitochondria stress test kit, according to manufacturer’s instructions. Sequential compound injections, including oligomycin A (1 μM), FCCP (1 μM) and Rotenone/antimycin A (0.5 μM), were applied on the microplate to test the glycolytic activity. The extracellular acidification rates (ECAR) were measured in real-time using a glycolysis stress test kit, according to manufacturer’s instructions. Sequential compound injections, including glucose (25 mM), oligomycin A (1 μM) and 2-DG (50 mM), were applied on the microplate to test the glycolytic activity.
6
Quantifying Cellular Glycolysis and OXPHOS
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To evaluate the glycolysis and OXPHOS levels, 661 W cells were plated onto Seahorse 96‐well plates. Cells were transfected with Hk2 siRNAs or negative control siRNAs before being washed and incubated in the base medium (Agilent Technologies) at 37°C for 1 h. ECAR and OCR were measured by glycolysis stress tests and mitochondria stress tests on seahorse Bioscience X96 extracellular flux analyzer (Agilent Technologies) according to the manufacturer's instructions, serving as quantitative indicators for cellular glycolysis and OXPHOS levels, respectively.34
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