The Seahorse XF96 Flux Analyzer (Seahorse Bioscience, Billerica, Massachusetts, USA) was used to measure the oxygen consumption rate (OCR) and extracellular acidification rate (ECAR) in lung cancer cells according to the manufacturer's instructions. Approximately 1×105 A549, H1299, XL-2, and H292 cells per well were seeded into an XF96-well plate and attached overnight. For the assessment of ECAR, cells were incubated with non-buffered RPMI 1640 under basal conditions followed by a sequential injection of 10 mM glucose, 1 mM mitochondrial poison (oligomycin, Sigma-Aldrich, Saint Louis, Missouri, USA) and 80 mM glycolysis inhibitor (2-deoxyglucose, 2-DG, Sigma-Aldrich). OCR was assessed under basal conditions and after sequential injection of 1 μM oligomycin, 1 μM fluoro-carbonyl cyanide phenylhydrazone (FCCP) and 2 mM antimycin A and rotenone (Sigma-Aldrich, Saint Louis, Missouri, USA). Both ECAR and OCR measurements were normalized to total protein content.
Seahorse xf96 flux analyzer
Manufactured by Agilent Technologies
Sourced in United States
The Seahorse XF96 Flux Analyzer is a laboratory instrument designed to measure the metabolic activity of cells. It can simultaneously measure the oxygen consumption rate and extracellular acidification rate of cells in a 96-well microplate format.
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Lab products found in correlation
Oligomycin, by Merck Group (10 mentions)
Antimycin a, by Merck Group (8 mentions)
Antimycin a and rotenone, by Merck Group (6 mentions)
2 deoxyglucose 2 dg, by Merck Group (5 mentions)
Rotenone, by Merck Group (5 mentions)
Carbonyl cyanide 4 trifluoromethoxy phenylhydrazone fccp, by Merck Group (5 mentions)
Xf assay medium, by Agilent Technologies (5 mentions)
Xf96 cell culture plate, by Agilent Technologies (3 mentions)
Fluoro carbonyl cyanide phenylhydrazone (fccp), by Merck Group (3 mentions)
Nanodrop nd 1000 spectrophotometer, by Thermo Fisher Scientific (3 mentions)
Xf base medium, by Agilent Technologies (2 mentions)
Oligomycin a, by Merck Group (2 mentions)
D glucose, by Merck Group (2 mentions)
Sodium pyruvate, by Merck Group (2 mentions)
Cell culture microplate, by Agilent Technologies (2 mentions)
Laminin 1, by Bio-Techne (2 mentions)
Standard assay kits, by Beyotime (2 mentions)
Mitochondrial stress test kit, by Agilent Technologies (2 mentions)
Seahorse xf cell mito stress test kit, by Agilent Technologies (2 mentions)
Rpmi 1640 medium, by Thermo Fisher Scientific (2 mentions)
49 protocols using seahorse xf96 flux analyzer
1
Metabolic Profiling of Lung Cancer Cells
2
Carnosine Modulates Cellular Bioenergetics
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The Seahorse XF96 Flux Analyzer (Seahorse Bioscience, Billerica, MA) was used to
determine the metabolic profiles of HeLa and SiHa cells under the influence of
carnosine. A total of 1.0 × 105 cells/well were seeded into XF96
microplates and incubated at 37°C for 24 hours. Thereafter, the cells were
treated for 48 hours with 20 mM carnosine. After treatment, the cells were
switched to unbuffered DMEM supplemented with 2 mM sodium pyruvate and 20 mM
carnosine 1 hour prior to the beginning of the assay and maintained at 37°C.
After baseline measurements, oxygen consumption rates (OCRs) and extracellular
acidification rates (ECARs) were measured after sequentially adding to each well
20 µL of oligomycin (which blocks the mitochondrial complex V, where the
electron chain is coupled to ATP synthesis), FCCP (an uncoupling agent that
allows maximum electron transport), and rotenone (which blocks complex I,
thereby eliminating mitochondrial respiration), to reach working concentrations
of 1 µg/mL, 1 µM, and 1 µM, respectively. Each parameter, including ATP-linked
OCR, proton leak, mitochondrial respiration OCR, and non-mitochondrial
respiration OCR, is derived as described previously.19 (link) OCR is reported in the unit of picomoles per minute, and ECAR is reported
in milli-pH units (mpH) per minute.
determine the metabolic profiles of HeLa and SiHa cells under the influence of
carnosine. A total of 1.0 × 105 cells/well were seeded into XF96
microplates and incubated at 37°C for 24 hours. Thereafter, the cells were
treated for 48 hours with 20 mM carnosine. After treatment, the cells were
switched to unbuffered DMEM supplemented with 2 mM sodium pyruvate and 20 mM
carnosine 1 hour prior to the beginning of the assay and maintained at 37°C.
After baseline measurements, oxygen consumption rates (OCRs) and extracellular
acidification rates (ECARs) were measured after sequentially adding to each well
20 µL of oligomycin (which blocks the mitochondrial complex V, where the
electron chain is coupled to ATP synthesis), FCCP (an uncoupling agent that
allows maximum electron transport), and rotenone (which blocks complex I,
thereby eliminating mitochondrial respiration), to reach working concentrations
of 1 µg/mL, 1 µM, and 1 µM, respectively. Each parameter, including ATP-linked
OCR, proton leak, mitochondrial respiration OCR, and non-mitochondrial
respiration OCR, is derived as described previously.19 (link) OCR is reported in the unit of picomoles per minute, and ECAR is reported
in milli-pH units (mpH) per minute.
3
Cellular Metabolic Profiling Using Seahorse
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The cellular oxygen consumption rate (OCR) and extracellular acidification rate (ECAR) were determined using a Seahorse™ XF96 Flux analyzer (Seahorse Bioscience, North Billerica, MA, USA). Experiments were performed according to the manufacturer’s instructions. Briefly, HEK293T cells were seeded in XF96 cell culture plates at 104 cells per well and transfected with increasing concentrations of TDP-43–6xHis plasmid. The XF96 sensor cartridges were hydrated with 200 μL calibrant pH 7.4 and stored overnight at 37 °C without CO2. On the day of analysis, the culture medium was replaced with XF DMEM medium, lacking bicarbonate (pH 7.4), and supplemented with glutamine (2 mM). Cells were then incubated at 37 °C in a non-CO2 incubator for 1 h. Sequential injection of glucose (10 mM), oligomycin (1 μM), dinitrophenol (DNP, 100 μM), or rotenone/antimycin A (0.5 mM) were added according to the supplier’s technical specifications to determine the principal metabolic parameters (Sigma-Aldrich). Specific mitochondrial and glycolytic ATP productions were assessed using Seahorse XF Real-Time ATP Rate Assay Kit (Seahorse Bioscience) according to the manufacturer protocol. Results were normalized in relation to DNA content per well using CyQuant dye (ThermoFisher Inc.).
4
Seahorse Analyzer for Oxygen Consumption
5
Extracellular Acidification Rate in Fibroblasts
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The assays for extracellular acidification rate (ECAR) in fibroblasts were performed with the Seahorse XF96 Flux Analyzer (Seahorse Bioscience, Agilent) according to the manufacturer’s instructions. Briefly, shNC or shSpry2 fibroblasts were seeded in a XF96-well plate at a density of 1 × 104 per well. The medium was replaced with assay media at 1 h before the assay. For the glycolytic stress test, 10 mM glucose, 1 μM oligomycin and 50 mM 2-deoxyglucose (2-DG) were injected to the wells. The measurements were normalized by total protein quantitation. Above experiments were performed in triplicate manner and repeated twice.
6
Measurement of ATP Production in BMDCs
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The ATP production rate was measured using a Seahorse XF-96 Flux Analyzer (Seahorse Biosciences, USA). The culture medium consisted of RPMI160 supplemented with 10% FBS and penicillin-streptomycin-amphotericin B. BMDCs were seeded in a XF-96 culture plate at a density of 1 × 105 cells/well and incubated overnight. The cells were treated with LPS 100 ng/ml with or without allithiamine 10 µM for 16 h. The assay medium comprised of XF base medium (Seahorse Biosciences) supplemented with 5.5 mM D-glucose (Sigma-Aldrich, USA), 1 mM sodium pyruvate (Sigma-Aldrich), and 1X GlutaMAXTM (Gibco, USA), adjusted to pH 7.4. The inhibitors were used in the following concentrations: oligomycin A (2 µmol/L; Sigma-Aldrich), rotenone (3 μmol/L; Sigma-Aldrich), and antimycin A (3 μmol/L; Sigma-Aldrich).
7
Mitochondrial Respiration Measurement in Hematological Cells
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OCR was assessed using a Seahorse XF96 flux analyzer (Seahorse Bioscience). KG1 (75 × 103 cells/well) and TEX (1 × 105 cells/well) cells were cultured in a 96-well XF96 culture plate (Seahorse Bioscience) coated with BD Cell-Tak (BD Biosciences) in their regular growth medium for 24 h with or without R406. An hour prior to the analysis, cells were washed with PBS and resuspended in XF base medium (pH 7.4) (Agilent Technologies) supplemented with glucose (5 mM), L-glutamine (1.6 mM), and pyruvate (1 mM). Plates were incubated for 1 h at 37 °C in a CO2-free incubator and transferred to the XF96 analyzer. OCR was measured at baseline and after the injection of oligomycin (1 µg/ml), FCCP (1.5 µM for TEX and 1.25 µM for KG1), rotenone/antimycin A (1 µM). After measurements, cells were washed with PBS, lysed in 10 μL of 0.1% Triton/PBS solution and frozen at −80 °C overnight. Thereafter, the protein concentration in each well was measured using BCA (Copper(II) sulfate solution and Bicinchoninic Acid solution, Sigma-Aldrich). Data were normalized to protein concentration and analyzed using Wave 2.0 software (Agilent Technologies).
8
Metabolic Profiling of Cancer Cells
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The assays for extracellular acidification rate (ECAR) and oxygen consumption rate (OCR) in the cultured cells were performed with the Seahorse XF96 Flux Analyzer (Seahorse Bioscience, Agilent) according to the manufacturer’s instructions. Briefly, OVCAR8 and ES-2 cells were seeded in a XF96-well plate at a density of 1 × 104 per well with indicated treatments. The media was replaced with assay media at 1 h before the assay. For the glycolytic stress test (Seahorse Cat. #103020-100), 10mM glucose, 1µM oligomycin and 50mM 2-deoxyglucose (2-DG) were injected to the wells. For the mitochondrial stress test (Seahorse Cat. #103015-100), 1µM oligomycin, 1µM FCCP, 0.5µM rotenone and 0.5µM antimycin A were added to the wells. Above experiments were performed in triplicate manner and repeated twice.
9
Metabolic Profiling of Cardiomyocytes
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The Seahorse XF-96 Flux Analyzer (Seahorse Bioscience Inc. Billerica, MA USA) was used to determine the metabolic profile of cardiomyocytes. 1.5×104 cells per well were seeded in Roswell Park Memorial Institute (RPMI) assay medium without sodium bicarbonate in a V3-PET cell culture plate. The cells were then treated with 400 µM Palmitate or BSA (kit provided by Seahorse Bioscience) for 30 minutes prior to mitochondrial stress test analysis. Titrations were performed to determine the optimal concentrations of: oligomycin (1 µM) (Sigma–Aldrich), carbonyl cyanide 4-trifluoromethoxy-phenylhydrazone (FCCP) (1 µM) (Sigma–Aldrich), and Antimycin A (10 µM) (Sigma–Aldrich). After analysis, cardiomyocytes were lysed in 0.1% Triton X-100 and 10 µM Tris-HCl and quantified using the Bradford Protein Assay (Biorad). The metabolic flux data were normalized to total protein and analyzed for Basal Respiration, ATP Production and Maximal Respiration rate.
10
Extracellular Flux Analysis of CRC Cells
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The Seahorse XF96 Flux Analyzer (Seahorse Bioscience, Agilent) was used to carry out the extracellular acidification rate (ECAR) and oxygen consumption rate (OCR) in the CRC cell lines. Briefly, all CRC cells used in this paper, including LoVo, RKO, SW620, and SW480 cells, were seeded into an XF96-well plate. The media were replaced with assay media 1 h before the assay. For ECAR assay (Seahorse Cat.#103020-100), 10 mM glucose, 1 μM oligomycin, and 50 mM 2-deoxyglucose (2-DG) were added to the wells. For the OCR test (Seahorse Cat.#103015-100), 1 μM oligomycin, 1 μM FCCP, 0.5 μM rotenone, and 0.5 μM actinomycin A were added to the wells at a special time point. Both measurements were normalized by total protein quantitation. The above experiments were performed in triplicate and repeated twice.
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