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Seahorse xfe24 bioanalyzer

Manufactured by Agilent Technologies
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The Seahorse XFe24 Bioanalyzer is a laboratory instrument designed to measure cellular metabolism and bioenergetics. It provides real-time analysis of oxygen consumption rate and extracellular acidification rate in live cells, allowing researchers to assess cellular respiratory function and glycolytic activity.

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Lab products found in correlation

Pyruvate, by Agilent Technologies (3 mentions) Xf plasma membrane permeabilizer guide pmp, by Agilent Technologies (2 mentions) Seahorse xfe24 cell culture microplate, by Agilent Technologies (2 mentions) Glutamine, by Agilent Technologies (2 mentions) Glucose, by Merck Group (2 mentions) Mitochondrial stress test kit, by Agilent Technologies (2 mentions) Xfe24 cell culture microplate, by Agilent Technologies (2 mentions) Enliten atp assay kit, by Promega (1 mentions) Microplate reader, by BMG Labtech (1 mentions) Seahorse xf cell mito stress test kit, by Agilent Technologies (1 mentions) Seahorse xfp, by Agilent Technologies (1 mentions) Seahorse xf assay media, by Agilent Technologies (1 mentions) Cell tak, by Corning (1 mentions) Lympholyte mammal, by Cedarlane (1 mentions) Low melting point agarose, by Thermo Fisher Scientific (1 mentions) Oligomycin, by Merck Group (1 mentions) Pyruvate, by Thermo Fisher Scientific (1 mentions) Xf base media, by Agilent Technologies (1 mentions) Xf24 cell culture microplate, by Agilent Technologies (1 mentions) 2 deoxy d glucose, by Merck Group (1 mentions)

22 protocols using seahorse xfe24 bioanalyzer

1

Measuring Mitochondrial Respiration in Cells

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Cells were plated and normalized as above and the XFe24 Seahorse Bioanalyzer was used to measure OCR. Activity was assessed in 1x MAS buffer (described in XF Plasma Membrane Permeabilizer Guide (PMP); Agilent) including 10 mM pyruvic acid, 2 mM malate, 4 μM FCCP, 4 mM ADP, 0.2% (w/v) fatty acid-free BSA and 1 nM PMP reagent. Final concentrations of injected reagents were: 2 μM rotenone, 10 mM succinate, 1.5 μg/ mL antimycin A, 10 mM ascorbate, and 100 μM tetramethyl-p-phenylenediamine (TMPD).
Garcia D., Carr J.F., Chan F., Peterson A.L., Ellis K.A., Scaffa A., Ghio A.J., Yao H, & Dennery P.A. (2020). Short exposure to hyperoxia causes cultured lung epithelial cell mitochondrial dysregulation and alveolar simplification in mice. Pediatric research, 90(1), 58-65.
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2

Measuring Mitochondrial Respiration in Cells

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Cells were plated and normalized as above and the XFe24 Seahorse Bioanalyzer was used to measure OCR. Activity was assessed in 1x MAS buffer (described in XF Plasma Membrane Permeabilizer Guide (PMP); Agilent) including 10 mM pyruvic acid, 2 mM malate, 4 μM FCCP, 4 mM ADP, 0.2% (w/v) fatty acid-free BSA and 1 nM PMP reagent. Final concentrations of injected reagents were: 2 μM rotenone, 10 mM succinate, 1.5 μg/ mL antimycin A, 10 mM ascorbate, and 100 μM tetramethyl-p-phenylenediamine (TMPD).
Garcia D., Carr J.F., Chan F., Peterson A.L., Ellis K.A., Scaffa A., Ghio A.J., Yao H, & Dennery P.A. (2020). Short exposure to hyperoxia causes cultured lung epithelial cell mitochondrial dysregulation and alveolar simplification in mice. Pediatric research, 90(1), 58-65.
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3

Measuring mitochondrial respiration

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Cells were plated and normalized as above and the XFe24 Seahorse Bioanalyzer was used to measure OCR. Activity was assessed in 1x MAS buffer (described in XF Plasma Membrane Permeabilizer (PMP) Guide; Agilent) including 10 mM pyruvic acid, 2 mM malate, 4 μM FCCP, 4 mM ADP, 0.2% (w/v) fatty acid-free BSA and 1 nM PMP reagent [41 (link)]. Final concentrations of injected reagents were: 2 μM rotenone, 10 mM succinate, 1.5 μg/mL antimycin A, 10 mM ascorbate, and 100 μM tetramethyl-p-phenylenediamine (TMPD).
Carr J.F., Garcia D., Scaffa A., Peterson A.L., Ghio A.J, & Dennery P.A. (2020). Heme Oxygenase-1 Supports Mitochondrial Energy Production and Electron Transport Chain Activity in Cultured Lung Epithelial Cells. International Journal of Molecular Sciences, 21(18), 6941.
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4

Measuring Cellular Oxidative Phosphorylation

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Oxidative phosphorylation was measured using the XFe24 Seahorse Bioanalyzer (Agilent) following manufacturer’s instructions. In the acute model, cells were seeded in seahorse assay plates 24 hours in air (21% O2/ 5 % CO2) prior to assay. The day of the assay, cells were exposed to hyperoxia for 4 hours. In the recovery model, cells were exposed to 4 hours of hyperoxia, trypsinized, counted, and seeded in Seahorse plates in air for 24 hours. Immediately prior to the assay cells were confirmed to be evenly seeded and one well from each condition was counted by trypan blue exclusion and used for normalization.
Garcia D., Carr J.F., Chan F., Peterson A.L., Ellis K.A., Scaffa A., Ghio A.J., Yao H, & Dennery P.A. (2020). Short exposure to hyperoxia causes cultured lung epithelial cell mitochondrial dysregulation and alveolar simplification in mice. Pediatric research, 90(1), 58-65.
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5

Measuring Cellular Oxidative Phosphorylation

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Oxidative phosphorylation was measured using the XFe24 Seahorse Bioanalyzer (Agilent) following manufacturer’s instructions. In the acute model, cells were seeded in seahorse assay plates 24 hours in air (21% O2/ 5 % CO2) prior to assay. The day of the assay, cells were exposed to hyperoxia for 4 hours. In the recovery model, cells were exposed to 4 hours of hyperoxia, trypsinized, counted, and seeded in Seahorse plates in air for 24 hours. Immediately prior to the assay cells were confirmed to be evenly seeded and one well from each condition was counted by trypan blue exclusion and used for normalization.
Garcia D., Carr J.F., Chan F., Peterson A.L., Ellis K.A., Scaffa A., Ghio A.J., Yao H, & Dennery P.A. (2020). Short exposure to hyperoxia causes cultured lung epithelial cell mitochondrial dysregulation and alveolar simplification in mice. Pediatric research, 90(1), 58-65.
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6

Quantifying Cellular Metabolic Activity

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Measurement of lactic acid was adapted from Brandt et al (1980) (link). In brief, appropriate amount of culture media was incubated at RT for 30 min in a final 100 μl of reaction mix containing 160 mM Tris–hydrazine, pH 9.0, 2.5 mM NAD+, 0.01% BSA, and 8 U lactate dehydrogenase. The amount of lactic acid was extrapolated from a standard curve based on the Ab340 reading recorded on a microplate reader (BMG LABTECH). Cellular ATP level was determined using ENLITEN ATP assay kit (Promega) according to the manufacturer’s specification. Intact cellular oxygen consumption was measured in the WT and ΔSHMT2 cells on an XFe24 seahorse bioanalyzer (Agilent) on plating 5 × 104 cells per well. Data have been represented as oxygen consumption relative to the WT cells.
Lucas S., Chen G., Aras S, & Wang J. (2018). Serine catabolism is essential to maintain mitochondrial respiration in mammalian cells. Life Science Alliance, 1(2), e201800036.
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7

Mitochondrial Function Assay in Adipocytes

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Primary preadipocytes were plated (20,000 cells/well) and differentiated in XF24 V7 PET cell culture microplates using the protocol described above. On the day of assay, cells were switched to XF-OCR assay media containing 2% free-fatty acid free BSA in the presence or absence of dibutyryl cAMP for four hours prior to the mito-stress assay (described in detail in the online supplement) using XFe24 Seahorse bioanalyzer (Agilent XF Seahorse).
For fatty acid oxidation (FAO) assay, cells were switched from growth media to substrate-limited media twenty four- hours prior to the assay. Forty five minutes prior to the assay, cells were switched to FAO media and incubated in the non-CO2 incubator at 37 °C. Etomoxir (40 μM) was added to the specified wells fifteen minutes prior to the assay. XF palmitate-BSA FAO (100 μM) substrate or BSA control were added right before running the mito-stress as per the protocol described in Rogers et al.70 (link).
Chechi K., Voisine P., Mathieu P., Laplante M., Bonnet S., Picard F., Joubert P, & Richard D. (2017). Functional characterization of the Ucp1-associated oxidative phenotype of human epicardial adipose tissue. Scientific Reports, 7, 15566.
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8

Adipocyte Mitochondrial Respiration Analysis

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The OCR of cultured adipocytes and adipose tissues was measured using Seahorse XF Cell Mito Stress Test Kit (Agilent) and analyzed by the XFe24 Seahorse bioanalyzer (Agilent). Adipocytes were treated with DHT (Meilunbio) or DMSO for 24 h and then equilibrated in carbonate free medium and incubator for 1 h. After measuring basal levels of OCR, the following drugs were sequentially loaded to each well: oligomycin (5 µm), FCCP (5 µm), rotenone (3 µm), antimycin (5 µm). 2 mg of adipose tissues were equilibrated at 37 °C in carbonate free condition for 1 h before measurement.
Zhao S., Nie T., Li L., Long Q., Gu P., Zhang Y., Sun W., Lin Z., Liu Q., Qi Y., Wang W., Xie M., Loomes K., Cai C., Wu D, & Hui H.X. (2023). Androgen Receptor is a Negative Regulator of PRDM16 in Beige Adipocyte. Advanced Science, 10(21), 2300070.
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9

Mitochondrial and Glycolytic Analysis

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Mitochondrial respiration and glycolytic function of WT and HO-1 knockout cells were measured in a XFe24 Seahorse Bioanalyzer and data were analyzed according to the manufacturer’s calculations (Agilent; Mitochondrial Stress Test and Glycolytic Rate Assay, Santa Clara, CA, USA). Cells were seeded the day prior to the assay. In order to account for biological batch to batch variability in cell growth overnight, immediately prior to the assay cells were confirmed to be evenly seeded and one well from each condition was counted by trypan blue exclusion and used for normalization. Final concentrations of injected reagents were: 1 μM oligomycin; 2 μM FCCP (carbonyl cyanide-4 (trifluoromethoxy) phenylhydrazone); 5 μM each rotenone and antimycin A; 10 mM glucose; and 50 mM 2-DG (2-deoxy-glucose).
Carr J.F., Garcia D., Scaffa A., Peterson A.L., Ghio A.J, & Dennery P.A. (2020). Heme Oxygenase-1 Supports Mitochondrial Energy Production and Electron Transport Chain Activity in Cultured Lung Epithelial Cells. International Journal of Molecular Sciences, 21(18), 6941.
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10

Mitochondrial Respiratory Chain Analysis

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The fundamental parameters of the mitochondrial respiratory chain (basal oxygen consumption rate (OCR), maximal OCR, ATP-linked respiration, spare respiratory capacity (SRC), and proton leak) were measured following 4 and 8 days of exposure to EtBr using the Seahorse XFe24 Bioanalyzer (Seahorse Bioscience, Massachusetts, USA) as previously described [32 (link), 33 (link)]. Briefly, 50 nematodes were loaded into each well of a Seahorse utility plate and 8 basal OCR readings were taken. Nematodes were then exposed to either 25μM FCCP (mitochondrial uncoupler), 20μM DCCD (ATP synthase inhibitor), or 10mM sodium azide (cytochrome c oxidase inhibitor) and an additional 8, 16, or 4 oxygen consumption measurements were taken, respectively. SRC was calculated by subtracting a wells basal OCR from its FCCP response, ATP-linked respiration was calculated by subtracting a wells DCCD response from its basal OCR, while proton leak was calculated by subtracting a wells azide response from its DCCD response. All experiments were repeated 3 separate times.
Luz A.L, & Meyer J.N. (2016). Effects of reduced mitochondrial DNA content on secondary mitochondrial toxicant exposure in Caenorhabditis elegans. Mitochondrion, 30, 255-264.
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