OPCs or pre-oligodendrcoytes were seeded onto PDL coated Seahorse cell culture plates (Agilent). The oxygen consumption rate was recorded using the manufacturers standard protocol for mitochondrial stress tests (Agilent). For the comparison of young and aged cells or OPCs and POLs, the cells were cultured overnight prior to the assay. For the comparison of aged cells treated with metformin and dorsomorphin the cells were cultured for 5d in the presence of the drugs. The cells were cultured 1h before the assay in modified assay medium (1.5mM sodium pyruvate, 2mM L-Glutamine, 2mM Glucose, 1% SATO in XF base medium, pH 7.4). The OCR measurements were carried out using a Seahorse XFp or a Seahorse XF96 analyzer. The basal OCR was calculated as the difference between the average of the measurements taken under untreated conditions and the average of the measurements taken after the injection of rotenone and antimycin A. All OCR values were then normalized to the cell number. The final OCR values were normalized to one treatment group and the results are represented as the relative basal oxygen consumption rate between the groups. The concentrations of the small molecules in the assay were: oligomycin (1μM), FCCP (0.5μM) rotenone (0.5μM) and antimycin A (0.5μM)
Seahorse cell culture plate
Manufactured by Agilent Technologies
Sourced in United States
Seahorse cell culture plates are specialized microplates designed for analyzing cellular metabolism and bioenergetics in a laboratory setting. They provide a platform for measuring key parameters such as oxygen consumption rate and extracellular acidification rate, which are indicators of cellular respiration and glycolysis, respectively.
Automatically generated - may contain errors
Lab products found in correlation
Un buffered dmem glutamine, by Agilent Technologies (2 mentions)
Sensor cartridge, by Agilent Technologies (2 mentions)
Xfe96 analyzer, by Agilent Technologies (1 mentions)
Rotenone, by Merck Group (1 mentions)
A2754, by Merck Group (1 mentions)
Seahorse xf analyzer, by Agilent Technologies (1 mentions)
Oligomycin, by Merck Group (1 mentions)
Fluoro carbonyl cyanide phenylhydrazone (fccp), by Merck Group (1 mentions)
Xfe96 cell culture microplate, by Agilent Technologies (1 mentions)
Seahorse extracellular flux analyzer, by Agilent Technologies (1 mentions)
Xf assay media, by Agilent Technologies (1 mentions)
In cell analyzer 6000 cell imaging system, by GE Healthcare (1 mentions)
In cell developer software, by GE Healthcare (1 mentions)
8 protocols using seahorse cell culture plate
1
Mitochondrial Respiration in Oligodendrocytes
2
Seahorse and Resipher Oxygen Assay
3
Seahorse Metabolic Profiling of H9c2 Cells
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Oxygen consumption was measured in H9c2 cells using a Seahorse XF Analyzer (Agilent Technology, Santa Clara, CA) as described before (Dott et al, 2014 (link)). H9c2 cells were seeded in Seahorse cell culture plates (Agilent Technology, Santa Clara, CA). Basal oxygen consumption rates (OCR) and extracellular acidification rates (ECAR) were measured to establish baseline rates. The cells were then treated with the following compounds in succession: 1 µg/mL oligomycin (Sigma, O4876), 0.5 µg/mL FCCP (Sigma, C2920), and 1 µg/mL rotenone (Sigma, R8875). Mitochondrial respirometry by Seahorse was conducted by an operator in a blinded fashion.
For the measurement of oxygen consumption in permeabilized cardiac fibers, left ventricular (LV) subendocardial muscle fibers were used to measure oxygen consumption rates and ATP synthesis as previously described (Boudina et al, 2005 (link)). Oxygen consumption rates were determined in the presence of palmitoyl-carnitine (Sigma, P1645) at 0.02 mM (VO), 1 mM ADP (A2754, Sigma) (VADP), and 1 μg/mL of the ATP synthase inhibitor oligomycin (A7699, Sigma) (VOligo). ATP levels were measured in fibers after the addition of 1 mM ADP.
For the measurement of oxygen consumption in permeabilized cardiac fibers, left ventricular (LV) subendocardial muscle fibers were used to measure oxygen consumption rates and ATP synthesis as previously described (Boudina et al, 2005 (link)). Oxygen consumption rates were determined in the presence of palmitoyl-carnitine (Sigma, P1645) at 0.02 mM (VO), 1 mM ADP (A2754, Sigma) (VADP), and 1 μg/mL of the ATP synthase inhibitor oligomycin (A7699, Sigma) (VOligo). ATP levels were measured in fibers after the addition of 1 mM ADP.
4
Seahorse Assay for Cellular Metabolism
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Collagen (Rat tail, type 1, Cat 354236, BD Biosciences)-coated Seahorse cell culture plates (Seahorse Bioscience, 102601-100) were seeded at a density of 20,000 cells per well (XFe96 cell culture microplate; Seahorse Biosciences, North Billerica, MA, USA). The cells were allowed to grow for 24–48 h at 37 °C in 5% CO2, after which the cells were washed and replaced with assay media (unbuffered DMEM or RPMI supplemented with 10 mM glucose, 1 mM sodium pyruvate, 2mM L-glutamine, no sodium bicarbonate at pH 7.4). The cells were incubated for 1 h at 37 °C in a non-CO2 incubator. Mitochondrial complex inhibitors (1.2 µM oligomycin, 1.2 µM carbonyl cyanide p-(trifluoromethoxyl)-phenyl-hydrazone (FCCP) and combined 1 µM rotenone with 1 µM antimycin A) were preloaded in the injection ports. For basal rate measurements, ECAR (Extra Cellular Acidification Rate) and OCR (Oxygen Consumption Rate) measurements were assessed. Experiments were performed in triplicates and the data were normalized by cell number.
5
Real-time Metabolic Profiling of Cells
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Cells were seeded at a density of ~ 1 × 105 cells per well in 24-well Seahorse cell culture plates (Seahorse Biosciences, North Billerica, MA, USA) in DMEM without carbonate. After incubation (1 h at 37 °C without CO2), real-time oxygen consumption rate (OCR) and extracellular acidification rate (ECAR) analyses were performed using Seahorse Extracellular Flux Analyzer (Seahorse Biosciences) as previously described by Barbier-Torres and coworkers [29 (link)].
6
Metabolic Analysis of Primary Myoblasts
7
Measuring Mitochondrial Respiration in Flp-In T-Rex 293 Cells
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Flp-In T-Rex 293 cells were induced in doxycycline (1 μg/ml) for 24 hours and then seeded (6 × 104 cells per well) in a 24-well Seahorse cell culture plate (100777-004, Seahorse Bioscience) precoated with Cell-Tak cell adhesive in 575 μl of XF assay media (101022-100, Seahorse Bioscience) supplemented with glucose (4.5 mg/ml), 2 mM l -glutamine, and 1× sodium pyruvate. Cells were incubated at 37°C with atmospheric CO2 for 1 hour. A XF24 flux plate prehydrated in calibrant buffer was loaded with 10 μM oligomycin, FCCP, and rotenone (final concentration, 1 μM) diluted in the supplemented XF assay medium. Three basal readings were taken (3 min each), followed by addition of oligomycin, FCCP, and rotenone, with three readings taken after the addition of each drug. Cells were washed with PBS once and then incubated in DMEM containing Hoechst 33342 (25 μg/ml) at 37°C for 30 min. The images were captured with an IN Cell Analyzer 6000 cell imaging system (GE Healthcare), and the nuclei number in each well was quantified using the IN Cell Developer software (GE Healthcare).
8
Metabolic Analysis of Primary Myoblasts
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Primary myoblasts were plated at a 1×104 cells/well of a collagen coated 96-well Seahorse cell culture plate and grown for 48hrs ± DCA in standard myoblasts growth media (+1g/L glucose, 1mM Pyruvate). As a negative control, one row of the culture plate was left without only media and no cells as a negative control. One day before measurements, a Seahorse sensor cartridge was prepared according to manufacturer’s instructions, with overnight incubation at 37°C in a CO2-free incubator. On the day of measurements, cells were washed twice and switched to un-buffered DMEM + Glutamine (Agilent) supplemented with 15% dialyzed fetal bovine serum (FBS) adjusted to pH of 7.4. For each condition (±DCA, pH 7.4), one row was supplemented with 1g/L glucose and 1mM pyruvate to assay respiration with available carbohydrate substrates, and one row left un-supplemented to assay non-carbohydrate respiration and lactate production. Cells were equilibrated at 37°C in a CO2-free incubator for one hour. Inhibitors (Oligomycin, Carbonyl cyanide-4-(trifluoromethoxy)phenylhydrazone (FCCP), and Rotenone/Antimycin) were added each at a final concentration of 1 μM after injection to the appropriate port of the sensor cartridge. The cell culture plate and sensor cartridge were run on the Seahorse XF-96, and data analyzed by Excel.
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