Primary or cultured podocytes were grown on 0.1 μg/ml Collagen I-coated plates in DMEM media supplemented with 5% FBS, 1% Antibiotic-Antimycotic. Cells were seeded onto a 96 well XFe96 microplate (part of Seahorse XFe96 FluxPak, Agilent) at a cell density of 3 × 104 cells/well, 2 days before assay to allow cells attachment. One day before the assay, cartridge plate (part of Seahorse XFe96 FluxPak, Agilent) was hydrated overnight with H2O and replaced with Calibrant (Agilent) for at least 1hr in a non-CO2 incubator. Cell plates were washed once with PBS and replaced with Seahorse Assay Medium (pH 7.4) (Agilent) and replaced again right before assay. Assay conditions and set up were performed using Mito Stress Test Kit and Glycolysis Stress Test Kit (Agilent) on a Seahorse XFe96 Analyzer (Agilent), according to manufacturer’s instructions. All Seahorse assay data was analyzed using Seahorse Wave v2.6.1 Software (Agilent).
Seahorse xfe96 fluxpak
Manufactured by Agilent Technologies
Sourced in Germany
The Seahorse XFe96 FluxPak is a specialized lab equipment designed for metabolic flux analysis. It provides a platform for real-time measurement of oxygen consumption rate and extracellular acidification rate in live cells. The FluxPak includes a sensor cartridge, calibration media, and consumables necessary for operating the Seahorse XFe96 Analyzer.
Automatically generated - may contain errors
Lab products found in correlation
Seahorse xfe96 analyzer, by Agilent Technologies (7 mentions)
Wave software, by Agilent Technologies (3 mentions)
Seahorse xf calibrant, by Agilent Technologies (2 mentions)
Xf cell culture microplate, by Agilent Technologies (2 mentions)
Oligomycin, by Merck Group (2 mentions)
Xf96 extracellular flux analyzer, by Agilent Technologies (2 mentions)
Mito stress test kit, by Agilent Technologies (2 mentions)
Calibrant, by Agilent Technologies (2 mentions)
Seahorse assay medium ph 7.4, by Agilent Technologies (2 mentions)
Seahorse wave v2, by Agilent Technologies (2 mentions)
Glycolysis stress test kit, by Agilent Technologies (2 mentions)
Bca protein assay, by Thermo Fisher Scientific (2 mentions)
Seahorse xfe96 analyser, by Agilent Technologies (1 mentions)
Fluoro carbonyl cyanide phenylhydrazone (fccp), by Merck Group (1 mentions)
Glucose, by Merck Group (1 mentions)
Ripa buffer, by Merck Group (1 mentions)
Complete protease inhibitor, by Roche (1 mentions)
Glutamine, by Merck Group (1 mentions)
Sodium pyruvate, by Merck Group (1 mentions)
Seahorse xf base medium, by Agilent Technologies (1 mentions)
14 protocols using seahorse xfe96 fluxpak
1
Podocyte Metabolic Profiling via Seahorse
2
Podocyte Metabolic Profiling via Seahorse
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Primary or cultured podocytes were grown on 0.1 μg/ml Collagen I-coated plates in DMEM media supplemented with 5% FBS, 1% Antibiotic-Antimycotic. Cells were seeded onto a 96 well XFe96 microplate (part of Seahorse XFe96 FluxPak, Agilent) at a cell density of 3 × 104 cells/well, 2 days before assay to allow cells attachment. One day before the assay, cartridge plate (part of Seahorse XFe96 FluxPak, Agilent) was hydrated overnight with H2O and replaced with Calibrant (Agilent) for at least 1hr in a non-CO2 incubator. Cell plates were washed once with PBS and replaced with Seahorse Assay Medium (pH 7.4) (Agilent) and replaced again right before assay. Assay conditions and set up were performed using Mito Stress Test Kit and Glycolysis Stress Test Kit (Agilent) on a Seahorse XFe96 Analyzer (Agilent), according to manufacturer’s instructions. All Seahorse assay data was analyzed using Seahorse Wave v2.6.1 Software (Agilent).
3
Bioenergetic Assessment of Bacterial Response to Drugs
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On the day prior to the assay the sensor cartridges from the Seahorse XFe96 FluxPaks (Agilent # 102416) were calibrated according to the manufacturer’s instructions using pre-warmed Seahorse XF Calibrant. E. coli were grown in LB medium overnight to and O.D. 600 of 0.3, washed in PBS and resuspended in Seahorse XF RPMI medium, pH 7.4 (Agilent # 103576) supplemented with 1% glucose. 105, 106 or 107 bacteria were added to XF Cell Culture Microplates (Agilent # 101085-004) precoated with poly-D-lysine and spin down at 2000g for 10 min to attach them to the plate. The wells in the plate were divided to include bacteria treated with TPP (negative control) and 3 concentrations (500, 100 & 20 μM) of C23.28-TPP; 8 technical replicates for each condition. 90 μl of fresh medium was added to each well and 90μl of TPP/prodrug solution was added to each injection port A. Baseline OCR and ECAR were measured for 12 min after which the TPP/prodrug solution was injected into each sample. Readings were taken as pmol/min (OCR) and mpH/min (ECAR) every 6 min for up to 90min. The mean of the 8 technical replicates was plotted for each treatment condition and changes in OCR and ECAR were compared to the control samples.
4
Bioenergetic Assessment of Bacterial Response to Drugs
5
Oxygen Consumption and Extracellular Acidification of Polarized Macrophages
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To analyse the extracellular acidification rate (ECAR) and oxygen consumption rate (OCR) of polarized macrophages, the Seahorse XFe96 FluxPak (Agilent, Waldbronn, Germany) was used as recommended by the manufacturer. CD14+ cells were isolated, and macrophages were polarized as previously described. Macrophages were polarized in the presence of 5, 10, or 50 μM sc1o. After polarization, cells were washed with Seahorse XF RPMI medium pH 7.4 (Agilent, Waldbronn) and incubated for 60 min at 37 °C. OCR and ECAR were measured for a total period of 160 min in the absence of sc1o. Cells were measured as octuplicates with 3 × 104 cells per well with the Seahorse XFe96 Analyser (Agilent, Waldbronn, Germany) and analysed with Wave Software (Agilent, Waldbronn, Germany). Values of vehicle (DMSO)-treated cells were set to 100% and sc1o-treated cells referred to this.
6
Seahorse Extracellular Flux Assay
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Experiments were performed according to the manufacture’s recommendations (Agilent, Santa Clara, CA, USA). Briefly, 5 × 104 BM-M were seeded per well (6–8 replicates) of an XF96 culture plate (Seahorse XFe96 FluxPak from Agilent) for 1 h at room temperature and then incubated overnight at 37°C with 5% CO2. Cells were treated as described in the results section. Cells were washed with warm PBS and then assay medium (sodium bicarbonate- and Glucose-free DMEM supplemented with sodium pyruvate 1 mM, pH 7.4) was added and the cells were kept for 1 h at 37°C without CO2. The background recording extracellular acidification rates (ECAR, milli pH/min) and oxygen consumption rates (OCR, pmol/min) were measured first. Glucose (25 mm; Sigma), Oligomycin (1,5 µM; Sigma), FCCP (1,5 µM; Sigma), and the mix of 2-deoxyGlucose (50 mM), Antimycin A (1 µM), and Rotenone (1 µM) were injected when indicated. After lysis of the cells with RIPA buffer (Merck) with proteinase inhibitors (Roche complete Protease Inhibitor, Basel, Switzerland) protein concentrations were determined using BCA protein assay from Thermo-Fischer Scientific (Waltham, MA, USA). ECAR and OCR data were normalized to protein concentration and analyzed as described before.21 (link)
7
Metabolic Profiling of Immune Cells
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For the analysis of the extracellular acidification rate (ECAR) and the oxygen consumption rate (OCR) for human monocytes, macrophages and dendritic cells, the Seahorse XFe96 FluxPak (Agilent) was used as recommended by the manufacturer. CD14+ cells were isolated and human monocytes were cultivated for 48 hours without further differentiation factors while macrophages and dendritic cells were differentiated as described before. All cells were cultivated in the presence of 5 nmol/L silvestrol during the experiment. After differentiation, cells were washed with Seahorse XF RPMI medium pH 7.4 (Agilent), incubated for 60 minutes at 37°C, and OCR and ECAR were measured for a total period of 160 minutes in the absence of silvestrol. Cells were stimulated after 30 minutes. Monocytes were stimulated with 100 ng/mL lipopolysaccharides (LPS) and 20 ng/mL IFN‐γ, macrophages were stimulated with 20 ng/mL IFN‐γ while dendritic cells were stimulated with a Stimulation‐Mix containing 5 ng/mL of human TNF‐α, IL‐6, IL‐1β and 500 ng/mL PGE2. Cells were measured as octuplicates (3 × 104 cells per well) using the Seahorse XFe96 Analyzer (Agilent) and analysed by Wave Software (Agilent).
8
Quantifying Mitochondrial Respiration in HAP1 Cells
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Oxygen consumption rate (OCR) of the HAP1 cells was quantified on a XF96 extracellular flux analyzer (Seahorse Bioscience) using the Seahorse XF Cell Mito Stress kit (Agilent, catalog number 103015-100) and according to the manufacturer’s protocol. The Seahorse XFe96 FluxPak (Agilent, catalog number 102601-100) and 50,000 HAP1 cells were plated in Seahorse base media (Seahorse XF base medium (Agilent, catalog number 102353-100) in 12 replicates per condition with 1% FCS, 1 mM glutamine (Sigma, catalog number 59202C-100ML) and 2 mM sodium pyruvate (Sigma, catalog number S8636-100ML). Plates were incubated in a CO2-free incubator at 37 °C for 1 h prior to acquisition on the Seahorse machine. Basal respiration (prior to oligomycin addition) and maximal respiration (following FCCP treatment) were calculated by subtracting non-mitochondrial oxygen consumption (values after rotenone & antimycin A addition).
9
Seahorse Assay for Mitochondrial Function
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Oxygen consumption rate (OCR) was measured using Seahorse XFe96 analyzer (Agilent, with Wave 2.3/4 software) and the Seahorse XFe96 FluxPak (102416-100, Agilent) according to the manufacturer’s protocols. Briefly, RAW/RAWAlox15 cells were seeded at 2.5 × 105 cells/well and peritoneal macrophages were seeded at 5 × 105 cells/well (and incubated with LPS (100 ng/ml)). Mitochondrial function was assessed using inhibitors (all from Sigma) injected in the following order: oligomycin (1 µM), FCCP (Carbonyl cyanide 4-(trifluoromethoxy)phenylhydrazone, 2 or 4 µM final concentration for peritoneal macrophages or RAW cells, respectively), rotenone (1 µM), antimycin A (10 μM for peritoneal macrophages; 1 µM for RAW/RAWAlox15 cells). OCR is described as pmol/min (per well).
10
Mitochondrial Function in Erythroid Cells
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Oxygen consumption rate (OCR) was measured on a Seahorse XFe96 Analyzer with a Seahorse XFe96 FluxPak (Agilent, Santa Clara, CA). Erythroid cells from BM of mice were isolated as previously described (Zhang et al., 2018 (link)). After recovery in IMDM with 10% FBS and 3 U/mL Epo at 37°C for 1 hr, 300,000 cells/well were seeded onto an XF96 cell culture microplate coated with Cell-tak (Corning, Corning, NY) and subjected to seahorse assay according to the protocol of the manufacturer. Three OCR measurements — basal, ATP-linked and maximal respiration — were performed after sequential injections of 1 µM oligomycin, 1 µM FCCP, and 0.5 µM Antimycin A/Rotenone. Five technical replicas from the same sample were performed for OCR measurement. Mitochondrial mass was determined by Mitotracker through flow cytometry analysis. The mitochondrial DNA (mtDNA) content was measured by qPCR as previously described (Liu et al., 2017 (link)). Briefly, after DNA isolation from the purified erythroid cells using a DNeasy Blood and Tissue Kit (Qiagen, Germantown), qPCR with specific primers for mtDNA and genomic DNA (gDNA) (Supplementary file 3a ) was performed to measure the ratio of mtDNA versus gDNA using the ΔΔCt method.
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