The Seahorse XF24 Analyzer was used as per the manufacturer's instructions (Seahorse Biosciences, North Billerica, MA) to measure the oxygen consumption rate (OCR) of cultured sympathetic neurons. Sympathetic neurons dissociated from the SCG of E21 rats were plated at a density of 15,000 cells/well onto 96-well plates specialized for use on the Seahorse XF24 Analyzer (Seahorse Biosciences) precoated with poly-D-lysine (100 μg/ml). Following the elimination of non-neuronal cells, cultures were treated with control media or BMP-7 at 50 ng/ml in the absence or presence of anti-oxidants for 48 h prior to measuring their oxygen consumption. At the end of the treatment period, neurons were incubated in DMEM with 5 mM glucose and 2 mM L-glutamine for 1 h at 37° C prior to loading them into the Seahorse analyzer. Following three measurements for baseline OCR in the first 35 min, the wells were injected with 100 μM 2,4-dinitrophenol (DNP) and three measurements for OCR were recorded in the next 35 min. Data are expressed as % of the first baseline OCR reading and presented as the mean ± SEM (n = 3 replicates per treatment group). Statistically significant differences between treatment groups were identified using one way ANOVA with post hoc Tukey's test.
Seahorse xf24 analyzer
Manufactured by Agilent Technologies
Sourced in United States, Canada
The Seahorse XF24 analyzer is a lab equipment product from Agilent Technologies. It is designed to measure the metabolic activity of cells in real-time, specifically their oxygen consumption rate (OCR) and extracellular acidification rate (ECAR).
Automatically generated - may contain errors
Lab products found in correlation
Oligomycin, by Merck Group (9 mentions)
Cell tak, by BD (8 mentions)
Rotenone, by Merck Group (6 mentions)
Xf cell mito stress test kit, by Agilent Technologies (6 mentions)
Oligomycin, by Agilent Technologies (5 mentions)
Antimycin a, by Merck Group (4 mentions)
Xf24 cell culture microplate, by Agilent Technologies (4 mentions)
2 deoxyglucose, by Merck Group (4 mentions)
Seahorse xf cell mito stress test kit, by Agilent Technologies (4 mentions)
Fluoro carbonyl cyanide phenylhydrazone (fccp), by Merck Group (3 mentions)
Xf cell culture microplate, by Agilent Technologies (3 mentions)
Glycolysis stress test kit, by Agilent Technologies (3 mentions)
Mito stress test, by Agilent Technologies (3 mentions)
Calibration buffer, by Agilent Technologies (2 mentions)
Rotenone, by Agilent Technologies (2 mentions)
Antimycin a, by Agilent Technologies (2 mentions)
Carbonyl cyanide 3 chlorophenylhydrazone cccp, by Merck Group (2 mentions)
Xf base medium, by Agilent Technologies (2 mentions)
Xf analyzer plate, by Agilent Technologies (2 mentions)
Glycolysis stress kit, by Agilent Technologies (2 mentions)
184 protocols using seahorse xf24 analyzer
1
Measuring Sympathetic Neuron Oxygen Consumption
2
Metabolic Profiling of Myotubes
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C2C12 myoblasts were seeded at 10,000 cells per well in 24-well XF plates and transfected with scramble or Dj1 siRNA. Myotubes were generated by replacing the medium with differentiation medium when cells reached confluency. After 4 days of differentiation, myotubes were switched to a bicarbonate-free medium and incubated for 1 h at 37 °C in a non-CO2 incubator. A Seahorse XF24 Analyzer (Seahorse Biosciences) was then used to measure the cellular bioenergetic profile. Each cycle included 3 min of mixing, a 2-min wait and measurement over 2 min. Three measurements were obtained at baseline and following injection of oligomycin (1 μM; Sigma), FCCP (0.5 μM; Sigma), and antimycin A and rotenone (1 μM; Sigma). Measurements were normalized to total protein content per well using the Bradford assay.
3
Measuring Mitochondrial Function Using Seahorse XF-24
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The OCR was measured in using a Seahorse XF-24 analyzer (Seahorse Bioscience). Cells were seeded at 20,000–50,000 cells per well 18 h before the analysis. On the day before the experiment, the sensor cartridge was placed into the calibration buffer supplied by Seahorse Bioscience and incubated at 37°C in a non-CO2 incubator. Immediately before measurement, cells were washed and incubated at 37°C with media lacking sodium bicarbonate. Three readings were taken after each addition of mitochondrial inhibitor, but before injection of the subsequent inhibitor. 2 µg/ml oligomycin, 5 µM CCCP, and 2 µM rotenone were used as mitochondrial inhibitors. The OCR was automatically calculated and recorded by the sensor cartridge and Seahorse XF-24 software.
4
Hepatocyte Mitochondrial Respiration Assay
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The mitochondrial oxygen consumption rate (OCR) was measured using a Seahorse XF-24 analyzer (Seahorse Bioscience Inc., North Billerica, MA, USA) in 24-well plates. Hepatocytes were seeded at 2 × 104 cells per well 24 h before the analysis. On the day before the OCR measurement, the sensor cartridge was placed into calibration buffer (Seahorse Bioscience) and incubated in a non-CO2 incubator at 37 °C. Hepatocytes were washed and incubated in DMEM without sodium bicarbonate. The medium and mitochondrial OXPHOS inhibitors were adjusted to pH 7.4 on the day of the OCR assay. The basal OCR was measured three times, and three readings were taken after the addition of each mitochondrial OXPHOS inhibitor [oligomycin (2 µg/mL) and rotenone (1 µM)]. The basal and post-oligomycin OCRs were calculated by averaging the last three measurements after maintaining a steady state. Coupled respiration was expressed as the percent decrease from basal respiration. Additionally, carbonyl cyanide m-chlorophenyl hydrazone (CCCP; 5 µM) was used to measure maximal mitochondrial respiration of the cells. OCR was automatically calculated and recorded by the sensor cartridge and the Seahorse XF-24 software.
5
Evaluating Mitochondrial Respiration Inhibitors
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Example 4
These compounds were next evaluated in a seahorse platform to test whether they inhibit pyruvate-dependent respiration as α-KB does. Mitochondria were isolated from mouse liver. The oxygen consumption rate (OCR) was measured using a Seahorse XF-24 Analyzer (Seahorse Bioscience). For the electron flow assay, 10 mM sodium pyruvate, and 2 mM malate were provided for complex I respiration. 10 mM succinate, and 10 mM/100 M ascorbate/tetra-methylphenylenediamine were provided as substrates for complex II and complex IV respectively. All compounds were incubated with mitochondria for 10 minutes before measurement. All of the compounds inhibited respiration when pyruvate was provided as a respiratory substrate, but had no inhibitory effects when substrates for complex II or complex IV were provided (
6
Metabolic Profiling of Adipocytes
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A Seahorse XF24 Analyzer (Seahorse Bioscience) was used to assess oxygen consumption and extracellular acidification rates. MPCs (40,000) were plated in each well 2 days before induction of adipogenesis by the above protocol. The mitochondrial stress test, glycolysis stress test, and the XF Palmitate-BSA FAO Substrate kits were used according to the manufacturer's protocol. Carbonyl cyanide m-chlorophenylhydrazone was used at 0.5 μM final concentration in the mitochondrial stress test, and at 2 μM final concentration in the FAO assay. Rate measurement cycles consisted of 2 min of mixing, 1 min of waiting, and 5 min of measurement.
7
Oxygen Consumption Measurement Protocol
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For oxygen consumption, 2 × 104 cells were plated and cultured for 24 hours. Next, oxygen consumption ratio (OCR) was measured adopting the Seahorse XF24 analyzer25 (Seahorse Bioscience, Santa Clara, CA, USA) under corresponding conditions, including basic condition and supplement of ATP synthase inhibitor (oligomycin), the mitochondrial uncoupler (carbonyl cyanide 4‐(trifluoromethoxy) phenylhydrazone; FCCP) and the complex I+II inhibitors (rotenone+antimycin A) following the manuals. All the chemical reagents were obtained from Sigma.
8
Measuring Glycolytic Capacity with Seahorse XF24
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We used a Seahorse XF24 analyzer (Seahorse Biosciences) to determine the glycolytic capacity according to the manufacturer’s instructions.
9
Mitochondrial Respiration Profiling in Thyroid Cells
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Oxygen consumption rate (OCR) was measured using a Seahorse XF-24 analyzer (Seahorse Bioscience Inc., North Billerica, MA, USA). Briefly, thyroid cells were seeded in 5 replicates in XF-24 plates (4 × 104 cells in 200 μL of growth medium per well) and placed in a 37 °C/5% CO2 incubator for 24 h. The sensor cartridge was placed into calibration buffer supplied by Seahorse Bioscience and incubated at 37 °C in a non-CO2 incubator for 24 h before the experiment. Immediately before measurement, the cells were washed and incubated in assay medium at 37 °C for 1 h in a non-CO2 incubator. Oligomycin A (20 µg/mL), an ATP synthase (complex V) inhibitor, was injected to measure cellular ATP production and carbonyl cyanide m-chloro phenyl hydrazine (optimized concentration, 50 μM). The uncoupling agent was used to measure maximal respiration by disrupting the mitochondrial membrane potential. Rotenone, a complex I inhibitor, was injected to inhibit mitochondrial respiration (20 μM), which allowed calculation of non-mitochondrial respiration. The OCR was automatically recorded by a sensor cartridge and calculated using Seahorse XF-24 software.
10
B Cell Metabolic Profiling by Seahorse
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