Mitochondrial oxygen consumption rate and extracellular acidification rate (ECAR) were measured in real time in live intact LCLs using a Seahorse Extracellular Flux (XF) 96 Analyzer (Agilent Technologies) with details previously published (9 (link)). Briefly, age- and gender-matched sets of LCLs consisting of ASD, Sib, and Con LCLs were seeded onto poly-d -lysine-coated 96-well XF-PS plates in XF DMEM with at least 3 replicate wells per treatment group. The cells were then treated for 1 h with the redox cycling agent DMNQ, which enters cells and generates both superoxide and hydrogen peroxide similar to levels generated by NADPH oxidase (11 (link)). DMNQ (5 mg/ml) was prepared in DMSO and diluted in XF DMEM into 10× stocks and added directly into wells of the XF plate incubated for 1 h at 37°C in a non-CO2 incubator with final concentrations of 5, 10, and 15 μM DMNQ. Previously, we demonstrated that these concentrations of DMNQ increase oxidative stress in LCLs (9 (link)).
Seahorse extracellular flux xf 96 analyzer
Manufactured by Agilent Technologies
Sourced in United States
The Seahorse Extracellular Flux (XF-96) analyzer is a lab equipment product manufactured by Agilent Technologies. It is designed to measure the metabolic activity of cells in real-time, providing data on oxygen consumption rate (OCR) and extracellular acidification rate (ECAR).
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Lab products found in correlation
Dmem xf assay media, by Agilent Technologies (1 mentions)
Glycolysis stress test, by Agilent Technologies (1 mentions)
Cell mitochondrial stress test, by Agilent Technologies (1 mentions)
Mixmate, by Eppendorf (1 mentions)
Nadp nadph glo assay, by Promega (1 mentions)
2 nbdg, by Cayman Chemical (1 mentions)
Xf96 well cell culture plates, by Agilent Technologies (1 mentions)
V7 microplate, by Agilent Technologies (1 mentions)
Oligomycin, by Merck Group (1 mentions)
Antimycin a, by Merck Group (1 mentions)
Rotenone, by Merck Group (1 mentions)
N acetylcysteine nac, by Merck Group (1 mentions)
Xf microplate, by Agilent Technologies (1 mentions)
Glucose assay kit, by Abcam (1 mentions)
Lactate assay kit, by Abcam (1 mentions)
Yo pro 1 assay, by Thermo Fisher Scientific (1 mentions)
25 protocols using seahorse extracellular flux xf 96 analyzer
1
Measuring Mitochondrial Function in LCLs
2
Extracellular Flux Analysis of Mitochondrial and Glycolytic Function
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The Seahorse Extracellular Flux XF96 Analyzer (Agilent) was used to measure oxygen consumption rate (OCR) and extracellular acidification rate (ECAR) in two assays, Cell Mitochondrial Stress Test (Agilent) and Glycolysis Stress Test (Agilent), following manufacturer instructions. Specifically, 2 × 104 KMRC2 or 786O cells per well were plated in quadruplicates in a 96-well XF culture plate in DMEM XF assay media (Agilent) supplemented with proper concentrations of glucose, sodium pyruvate and L-glutamine. For Mitochondrial Stress Test, oligomycin at 1.5 µM, FCCP at 1 µM and Rotenone/Antimycin A at 0.5 µM were optimized concentrations. For Glycolysis Stress Test, saturating glucose solution at 25 mM, oligomycin at 1 µM and 2-deoxy-glucose (2-DG) at 50 mM were optimized concentrations. After the assay, OCR and ECAR readings were normalized by crystal violet absorbance readings, which are a proxy for total cell content in each well. Cells were fixed and stained with 0.2% crystal violet/25% methanol solution for 10 min at room temperature. Excess stains were washed with PBS and ddH2O. SDS (1%) was added to solubilize crystal violet stain, following by shaking the plate for 5 min at 350 rpm on MixMate (Eppendorf). Finally, the absorbance was read at 570 nm subtracting the background absorbance at 690 nm.
3
Metabolic Profiling of Cellular Processes
4
Seahorse Extracellular Flux Analysis of Basal OCR in MCF7 Cell Lines
5
Extracellular Flux Analysis of Cellular Bioenergetics
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Real-time oxygen consumption rates (OCR) and extracellular acidification rates (ECAR) measurements were determined using the Seahorse Extracellular Flux (XF96) analyzer (Seahorse Bioscience, MA, USA). 1 × 104 cells per well were seeded into XFe-96 well cell culture plates, and incubated overnight to allow attachment. Control cells were processed in parallel. After 48 hours of incubation, cells were washed in pre-warmed XF assay media (or for OCR measurement, XF assay media supplemented with 10mM glucose, 1mM Pyruvate, 2mM L-glutamine and adjusted at 7.4 pH). Cells were then maintained in 175 μL/well of XF assay media at 37°C, in a non-CO2 incubator for 1 hour. During the incubation time, we loaded 25 μL of 80mM glucose, 9μM oligomycin, and 1M 2-deoxyglucose (for ECAR measurement) or 10μM oligomycin, 9μM FCCP, 10μM rotenone, 10μM antimycin A (for OCR measurement), in XF assay media into the injection ports in the XFe-96 sensor cartridge. Data sets were analyzed by XF96 software and GraphPad Prism software, using one-way ANOVA and Student's t-test calculations. All experiments were performed in quintuplicate, three times independently.
6
Seahorse Flux Analysis of Mitochondrial Function
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A Seahorse Extracellular
Flux (XF) 96 Analyzer (Seahorse Bioscience, Inc., North Billerica,
MA) was used to measure the oxygen consumption rate (OCR) and extracellular
acidification rate (ECAR) in C2C12 mouse myotubes. The OCR and ECAR
were measured after the cells were incubated for 18 h with compounds2 , 3 , and 6 (5, 10, and 20 μg/mL),
and the cells were washed with an XF base medium supplemented with
10 mM glucose, 2 mM glutamine, and 1 mM pyruvate (pH = 7.4) and were
then incubated in this medium for 1 h at 37 °C in a non-CO2 incubator. The plates were put in a Seahorse XF96 at 37 °C
for 10 min calibration and 3 measurement cycles to record basal cellular
respiration. Oligomycin (2 μM), FCCP (0.5 μM), and a mixture
of rotenone plus antimycin A (0.5 μM) were added sequentially,
and three measurements were performed after the addition of each concentration
of the compounds.44 (link)−46 (link)
Flux (XF) 96 Analyzer (Seahorse Bioscience, Inc., North Billerica,
MA) was used to measure the oxygen consumption rate (OCR) and extracellular
acidification rate (ECAR) in C2C12 mouse myotubes. The OCR and ECAR
were measured after the cells were incubated for 18 h with compounds
and the cells were washed with an XF base medium supplemented with
10 mM glucose, 2 mM glutamine, and 1 mM pyruvate (pH = 7.4) and were
then incubated in this medium for 1 h at 37 °C in a non-CO2 incubator. The plates were put in a Seahorse XF96 at 37 °C
for 10 min calibration and 3 measurement cycles to record basal cellular
respiration. Oligomycin (2 μM), FCCP (0.5 μM), and a mixture
of rotenone plus antimycin A (0.5 μM) were added sequentially,
and three measurements were performed after the addition of each concentration
of the compounds.44 (link)−46 (link)
7
Oxygen Consumption and ATP Activity in Hematopoietic Stem Cells
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Oxygen consumption in cells was determined using the Seahorse Extracellular Flux (XF-96) analyzer (Seahorse Bioscience, Chicopee, MA, USA). Bone marrow cells were isolated and treated with MnP at 20 μM for 2 h. The cells were then harvested, antibody-labeled and flow cytometry sorted in ice or at 4 °C. 200,000 LSK cells in triplicate for treatment or control were cytospinned to the bottom of a XF96 Tissue Culture Plate (Seahorse Bioscience) coated with BD Cell-Tak Cell Adhesive. OCR was measured three times and plotted as a function of cells under the basal condition followed by the sequential addition of oligomycin (1 μg/ml), FCCP (1 μM), or antimycin (2 μM) and rotenone (1 μM) for the ATP-linked, maximum, and reserve capacities of oxygen consumption, respectively.
BMNCs were isolated from mice (n=6). The cells were treated with either vehicle or 20 μM MnP for 1 h. at 37 °C, stained and sorted for LSK and MyPro (Lineage negative, c-kit positive, Sca1 negative) cells in ice or at 4 °C. Sorted cells (3×104) were lysed, and intracellular ATP activity was measured using the Luciferase ATP Bioluminescent somatic cell assay kit (Sigma, Cat# FLASC) following the manufacturer's instructions.
BMNCs were isolated from mice (n=6). The cells were treated with either vehicle or 20 μM MnP for 1 h. at 37 °C, stained and sorted for LSK and MyPro (Lineage negative, c-kit positive, Sca1 negative) cells in ice or at 4 °C. Sorted cells (3×104) were lysed, and intracellular ATP activity was measured using the Luciferase ATP Bioluminescent somatic cell assay kit (Sigma, Cat# FLASC) following the manufacturer's instructions.
8
Oxygen Consumption in H295R Cells
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Real-time oxygen consumption rates (OCR) were determined using the Seahorse Extracellular Flux (XF96) analyzer (Seahorse Bioscience, Billerica, MA, USA). H295R cancer cells were seeded into XF96-well cell culture plates (Seahorse Bioscience, Billerica, MA, USA) and incubated overnight at 37 °C in a 5% CO2 humidified atmosphere. After 48 h, cells were treated with curcumin (10, 20 μM) for 16 h. At the end of treatment, cells were processed as previously described [3 (link)]. The obtained OCR values were normalized to the protein content within the individual wells.
9
Mitochondrial Respiration Profiling in LCLs
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We used the state-of-the-art Seahorse Extracellular Flux (XF) 96 Analyzer (Seahorse Bioscience, North Billerica, MA, USA) to measure oxygen consumption rate, an indicator of mitochondrial respiration, in real-time in live intact LCLs. Each run contained the three matched samples (control, AD-N and AD-A) on the same plate. The Seahorse assay (Supplementary Figure 1A ), described previously,67 (link), 68 (link) provides measures of ATP-linked respiration, proton leak respiration, maximal respiratory capacity and reserve capacity.
10
Measuring Cellular Respiration in Breast Cancer Cells
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OCR and PPR for MCF7, MCF7/Mock and MCF7/PMA1/Clone 1 cells were measured by using the Seahorse Extracellular Flux (XF-96) analyzer (Seahorse Bioscience). The cells were cultured for one hour in the absence of glucose in a non-CO2 incubator prior to the measurements. Then the PPR and OCR were measured in the absence of glucose associated with non-glycolytic activity, following three sequential injections of D-glucose (5 mM), oligomycin (1 μM), and 2-deoxyglucose (50 mM) in real time. Glycolytic PPR was determined by the difference between the PPR in the presence of glucose and in the absence of glucose. Protein concentration was determined for each well using a standard BCA protein assay. The OCR and PPR values are normalized to μg protein.
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