Seahorse respirometry was performed with Min6 cells using an Agilent seahorse XF96e analyzer. Cellular and drug titration optimization experiments were performed to ensure maximal responses. Cells were incubated for 2 h prior to the experiment in substrate limited media, followed by Seahorse base media containing 3 mM glucose one hour prior to the initiation of recordings. Cellular stimulation was induced by glucose (16.7 mM), Oligomycin (1 µM), FCCP (1 µM), and rotenone/antimycin A (R/A; 1 µM). Protein determination was assessed at the end of the experiment (ThermoFisher) to normalize recordings. Analysis was performed off-line using Wave 2.6.1 software. Coupling efficiency was calculated using pre-set Wave calculations in normalized recordings, that is coupling efficiency was the ratio of ATP-driven respiratory rates over baseline respiratory rates (rate 3) × 100. Proton leak was calculated based on the difference between non-mitochondrial OCR (Post-R/A) and non ATP-synthase dependent OCR (Post-Oligo) respiration rates. No wells were excluded from the data presented.
Seahorse xf96e analyzer
Manufactured by Agilent Technologies
Sourced in United States
The Seahorse XF96e analyzer is a laboratory instrument designed for the measurement of cellular metabolism. It provides real-time, quantitative analysis of oxygen consumption rate (OCR) and extracellular acidification rate (ECAR) in a 96-well microplate format.
Automatically generated - may contain errors
Lab products found in correlation
G7528, by Merck Group (1 mentions)
Glucose uptake colorimetric assay kit, by Applygen (1 mentions)
Lactate assay kit, by Merck Group (1 mentions)
Seahorse xf glycolysis stress test kit, by Agilent Technologies (1 mentions)
L lactate assay kit, by Abcam (1 mentions)
Glucose test kit, by Applygen (1 mentions)
11 protocols using seahorse xf96e analyzer
1
Seahorse Respirometry of Min6 Cells
2
Mitochondrial Function Evaluation using Seahorse
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Mitochondrial function was assessed using the Seahorse XF96e Analyzer (Agilent Technologies, Santa Clara, CA). Following treatment, 150,000 cells per well were seeded on Cell-Tak coated Seahorse plates. Cells were equilibrated in XF media prior to measuring the mitochondrial oxygen consumption rate (OCR) and extracellular acidification rate (ECAR). The mitochondrial stress test was implemented as previously described [20] . Oligomycin (0.5 µg/ml), FCCP (0.6 µM), and antimycin A (10 µM) injections defined the following parameters: basal OCR, ATP-linked OCR, proton leak OCR, maximal OCR, reserve capacity, and non-mitochondrial OCR [21] (link). In addition, the oligo-sensitive ECAR was determined.
3
Mitochondrial Activity in hiPS-CMs
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Mitochondrial activity in hiPS-CMs was measured using a Seahorse XF96e analyzer (Agilent) [40 (link)]. hiPSC-CMs were plated at 20k per well in a 96-well XF plate. XF media was prepared using phenol red- and sodium bicarbonate-free DMEM (Corning 90-113-PB) supplemented with 11 mM glucose (Sigma Aldrich G7528), 1 mM pyruvate (Sigma Aldrich P5280), and 4 mM glutamine (Corning 61-030-RO). The basal oxygen consumption rate was normalized to protein content.
4
Glycolytic Activity Profiling
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The glucose uptake and lactate product were detected using the Glucose Uptake Colorimetric Assay Kit (Applygen Technologies, Beijing, China) and Lactate Assay Kit (#MAK064, Sigma, Shanghai, China) according to the manufacturer's instructions. The ECAR was determined using the Seahorse XF Glycolysis Stress Test Kit on a Seahorse XF96e analyzer (Agilent, Santa Clara, CA, USA) according to the manufacturer's instructions. Results were normalized to the cell numbers of each reaction. Experiments were performed in triplicate and repeated three times.
5
Measurement of Cellular Glycolytic Activity
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Measurement of cellular glycolytic activity was performed using the Seahorse XF96e analyzer (Agilent Technologies, Massachusetts, USA). N/TERT-HPV8-E7, N/TERT-HPV16-E7 and control cells were grown in RM + medium and seeded out on Seahorse XF96 cell culture microplates 16 h prior to measurement at a cell density of 25,000 cells per well. Assay medium was prepared on the day of the assay by supplementing Seahorse XF Base Medium with 2 mM glutamate and no other additives. Then the medium was warmed to 37 °C and the pH adjusted to 7.4. Forty-five minutes prior to measurement cell culture medium was replaced by assay medium, followed by adding 5 mM glucose, 1 μM Oligomycin and 100 mM 2-Deoxy-D-glucose (2-DG) at different time-points. Following measurement of glycolytic activity protein concentration was determined including a standard curve. All cell lines were at least measured n = 10 times and normalized to total protein content, including a BSA standard with known protein concentrations. The data was later analysed using the Seahorse Report Generator.
6
Glycolysis Stress Test in HT1080 Cells
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Measurements of ECAR in HT1080 cells were performed using the Seahorse XF96e analyzer (Seahorse Bioscience, North Billerica, MA, USA) according to the manufacter’s instruction. Briefly, 1.0 × 104 cells were seeded per well overnight in a 96-well XF cell culture microplate in growth medium. ECAR was measured with an XF96 analyzer in XF base medium containing 4 mM glutamine (pH 7.35) following sequential additions of glucose (10 μM), oligomycin (1 μM) and 2-DG (50 mM). Data were analysed by a Seahorse XF Glycolysis Stress Test Report Generator.
7
Cellular Bioenergetics Profiling
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Measurements of OCR and ECAR were performed using the Seahorse XF96e analyzer (Seahorse Bioscience) as described previously (Hall et al., 2013 ). Briefly, BJ or U2‐OS cells (1.0 × 104 cells/well) were seeded in XF96 V3 culture microplates (Seahorse Bioscience) one day before the experiment. OCR and ECAR were analyzed in the Seahorse assay buffer (containing 10 mM glucose, 10 mM pyruvate, pH 7.4).
8
Evaluation of Glucose Metabolism
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The glucose metabolism was evaluated by glucose uptake assay and extracellular acidification rate (ECAR) according to previous reports (Li et al., 2017 (link)). The glucose uptake was examined using the glucose test kit (Applygen Technologies) according to the manufacturer's instructions. The lactate product assay was performed using the l ‐lactate assay kit (BioVision) according to the manufacturer's instructions. The ECAR was measured in GC cells following transfections using the Seahorse XF96e analyzer (Seahorse Bioscience) according to the manufacture's instruction. ECAR of GC cells was measured in XF base medium containing 4 mM glutamine following the addition of 10 µM glucose, 1 µM oligomycin, and 50 mM 2‐DG. Experiments were repeated three times. Results were normalized by the ratio of the cell number of treated cells to that of control cells.
9
Glycolytic Capacity Profiling via Seahorse
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ECAR assay was determined by Seahorse XF96e analyzer (Seahorse Bioscience, USA) according to the manufacturer’s instructions. Briefly, 1.0 × 104 cells were plated per well in a 24-well XF cell culture microplate. ECAR was measured in XF base medium containing 4 mM glutamine (pH 7.35) following sequential additions of glucose (10 mM), oligomycin (1 mM) and 2-DG (50 mM). Data were analyzed by a Seahorse XF Glycolysis Stress Test Report Generator.
10
Glycolysis Stress Test in MCF-7 Cells
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The ECAR was measured in MCF-7 cells following lentiviral transduction and/or miRNA transfection using the Seahorse XF96e analyzer (Seahorse Bioscience, North Billerica, USA) according to the manufacture's instruction. Briefly, MCF-7 cells were seeded in a 96-well XF cell culture microplate with complete growth medium following lentiviral transduction and/or miRNA transfection. ECAR was measured using an XF96 analyzer in XF base medium containing 4 mM glutamine following sequential addition of 10 µM glucose, 1 µM oligomycin and 50 mM 2-DG. Data were analyzed by a Seahorse XF Glycolysis Stress Test Report Generator.
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