Seahorse xf96

Manufactured by Agilent Technologies

Sourced in United States

The Seahorse XF96 is a lab equipment product from Agilent Technologies. It is designed to measure cellular bioenergetics, including oxygen consumption rate and extracellular acidification rate, in a 96-well plate format.

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83 protocols using seahorse xf96

Measuring Oxygen Consumption in Cybrid Cells

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The oxygen consumption rates (OCRs) in various cybrid cell lines were measured with a Seahorse XF96 extracellular flux analyzer (Seahorse Bioscience, North Billerica, MA, USA), as detailed previously.³⁸ (link) Cybrid cells were seeded at a density of 2 × 10⁴ cells per well on the Seahorse XF96 polystyrene tissue culture plates. Inhibitors were used at the following concentrations: oligomycin (1.5 µM), carbonyl cyanide 4-(trifluoromethoxy)phenylhydrazone (FCCP; 0.8 µM), antimycin A (1.5 µM), and rotenone (3 µM).

Zhang J., Ji Y., Chen J., Xu M., Wang G., Ci X., Lin B., Mo J.Q., Zhou X, & Guan M.X. (2021). Assocation Between Leber's Hereditary Optic Neuropathy and MT-ND1 3460G>A Mutation-Induced Alterations in Mitochondrial Function, Apoptosis, and Mitophagy. Investigative Ophthalmology & Visual Science, 62(9), 38.

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Measuring Cellular Bioenergetics Using Seahorse XF96

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An XF96 seahorse (Seahorse Bioscience, MA, USA) was applied to detect the ATP rate of PMs. A total of 20,000 cells/well which had been transferred and seeded into the Seahorse XF96 culturing plates within medium. Wells divided into different treatment groups. Next day, cells were gently washed once in PBS and then cultured for one hour at 37 °C in Seahorse incubation medium. To ensure accurate detection of extracellular pH, cells were cultured in a CO₂-free incubator. The detection of OCR and extracellular acidification rate (ECAR) were performed at baseline and following sequential injections of oligomycin (2.5 μM), rotenone (1μM) and antimycin-A (1μM). All data were automatically calculated by the Seahorse XF96 software (Wave, Seahorse Bioscience, MA, USA).

Jin G., Guo N., Liu Y., Zhang L., Chen L., Dong T., Liu W., Zhang X., Jiang Y., Lv G., Zhao F., Liu W., Hei Z., Yang Y, & Ou J. (2023). 5-aminolevulinate and CHIL3/CHI3L1 treatment amid ischemia aids liver metabolism and reduces ischemia-reperfusion injury. Theranostics, 13(14), 4802-4820.

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Measuring Mitochondrial Activity in C. elegans

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Oxygen consumption rate (OCR) was assessed using the Seahorse XF96 (Seahorse Bioscience), following the protocol outlined in (Koopman et al., 2016 (link)). Briefly, a synchronized culture of ~100 worms was harvested on day 1 of adulthood with sterile M9 buffer. After three washes in the M9 buffer, the worms were transferred to a 96-well Seahorse plate, where their OCR was measured six times to determine mitochondrial activity for each condition at basal level and another six times measurement after adding 10 μM FCCP as the final concentration.

Gao A.W., Alam G.E., Zhu Y., Li W., Katsyuba E., Sulc J., Li T.Y., Li X., Overmyer K.A., Lalou A., Mouchiroud L., Sleiman M.B., Cornaglia M., Morel J.D., Houtkooper R.H., Coon J.J, & Auwerx J. (2024). High-content phenotypic analysis of a C. elegans recombinant inbred population identifies genetic and molecular regulators of lifespan. bioRxiv.

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Measuring Cellular Oxygen Consumption

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The oxygen consumption rate (OCR) was analyzed by a Seahorse XF96 extracellular flux analyzer (Seahorse Bioscience, Agilent, TX, USA). HUVECs (10,000 cells per well) were seeded in XFe96 Cell Culture Microplates (101085-004, Agilent), then incubated at 37 °C with 5% CO₂ for 24 h. After stabilization of the cellular metabolism, the cells were treated with or without 250 nM celastrol in EGM2 for 24 h. The subsequent experimental operations were performed in strict accordance with standard experimental procedures. Briefly, HUVECs were equilibrated in fresh XF DMEM base medium (103575-100, Agilent) containing 25 mM glucose (103577-100, Agilent), 1 mM sodium pyruvate (103578-100, Agilent), and 2 mM L-glutamine (103579-100, Agilent) for 1 h at a 37 °C incubator without extra CO₂. For the Mito Stress test, 1.5 μM oligomycin A (Oligo), 4 μM carbonyl cyanide-p-trifluoromethoxy-phenylhydrazone (FCCP), and 0.5 μM rotenone/antimycin A (A/R) were sequentially injected into each well operated by the instrument.

Li G., Zhou L., Deng H., Huang C., Wang N., Yue L., Zhang P., Zhou Y., Zhou W, & Gao Y. (2022). Targeting OPA1-Mediated Mitochondrial Fusion Contributed to Celastrol’s Anti-Tumor Angiogenesis Effect. Pharmaceutics, 15(1), 48.

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Measuring Oxygen Consumption in Cells

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Cells were plated in a Seahorse Biosciences 96‐well plate and allowed to adhere for 20 h. They were then treated with 20 μM C₁₈‐pyr‐cer or equivalent amount of vehicle (EtOH) for 2 h, and then, oxygen consumption rate was measured using a Seahorse XF96 (Seahorse Biosciences) as described by the manufacturer (Sentelle et al, 2012).

Thomas R.J., Oleinik N., Panneer Selvam S., Vaena S.G., Dany M., Nganga R.N., Depalma R., Baron K.D., Kim J., Szulc Z.M, & Ogretmen B. (2017). HPV/E7 induces chemotherapy‐mediated tumor suppression by ceramide‐dependent mitophagy. EMBO Molecular Medicine, 9(8), 1030-1051.

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Oxygen Consumption in C. elegans

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N2 and eat-2(ad1116) worms were grown on ev or sfa-1 RNAi bacteria from egg hatch. Worms were transferred to plates with FUDR on day 1 of adulthood. Oxygen consumption was measured on day 4 and day 15 of adulthood using a Seahorse XF96 analyser (Seahorse Bioscience). Worms were removed from NGM plates, washed 3 times with M9 buffer and transferred to a 96 well plate (10 worms/well). Basal respiration was measured 10 times followed by the addition of FCCP (10μM) to measure maximal respiration, which was measured 6 times. Oxygen consumption rates were normalized to the number of worms per well. Two biological replicate experiments were run.

Heintz C., Doktor T.K., Lanjuin A., Escoubas C., Zhang Y., Weir H.J., Dutta S., Silva-García C.G., Bruun G.H., Morantte I., Hoxhaj G., Manning B.D., Andresen B.S, & Mair W.B. (2016). Splicing Factor 1 Modulates Dietary Restriction and TORC1 Pathway Longevity in C. elegans. Nature, 541(7635), 102-106.

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Basal Oxygen Consumption in C. elegans

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Basal oxygen consumption was measured per previous methods66 (link) using a Seahorse XF96 instrument (Seahorse Bioscience Inc.). In brief, around 200 L4 nematodes/group were transferred to designated drug plates, followed by OCR detection on adult days 1 or 4. On the day of the experiment, all the C. elegans were collected using M9 buffer washing, followed by washing with M9 buffer twice. Nematodes were then re-suspended and transferred in 96-well standard Seahorse plates (#100777–004) (10~15 worms per well), and basal OCR was measured six times. After the experiments, nematodes numbers were counted, followed by data normalization to the number of nematodes in each individual well. We had ten replicates for each group, and repeated the experiment twice.

Fang E.F., Waltz T.B., Kassahun H., Lu Q., Kerr J.S., Morevati M., Fivenson E.M., Wollman B.N., Marosi K., Wilson M.A., Iser W.B., Eckley D.M., Zhang Y., Lehrmann E., Goldberg I.G., Scheibye-Knudsen M., Mattson M.P., Nilsen H., Bohr V.A, & Becker K.G. (2017). Tomatidine enhances lifespan and healthspan in C. elegans through mitophagy induction via the SKN-1/Nrf2 pathway. Scientific Reports, 7, 46208.

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Measuring Mitochondrial Respiration in C. elegans

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Oxygen consumption rate (OCR) was measured with the Seahorse XF96 (Seahorse Bioscience) as described (Koopman et al., 2016 (link)). In brief, ∼100 N2 and CB4856 worms were cultured on plates with or without Dox, and washed off with sterile M9 buffer after worms reached day 1 adulthood. After washing with M9 buffer for three times, worms were then transferred in a 96-well Seahorse plate and OCR was measured for six times. Mitochondrial OCR was measured for each condition.

Gao A.W., El Alam G., Lalou A., Li T.Y., Molenaars M., Zhu Y., Overmyer K.A., Shishkova E., Hof K., Bou Sleiman M., Houtkooper R.H., Coon J.J, & Auwerx J. (2022). Multi-omics analysis identifies essential regulators of mitochondrial stress response in two wild-type C. elegans strains. iScience, 25(2), 103734.

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Adipocyte Metabolic Profiling using Seahorse

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Oxygen consumption rates (OCRs) and extracellular acidification rates (ECARs) were measured using the Seahorse xF96 (Seahorse Bioscience). Adipocytes were grown in Seahorse cell culture plates as above. On day 7, adipocytes were washed twice in XF assay medium (Seahorse Bioscience) supplemented with 25‐mM glucose, 0.5‐mM sodium pyruvate, 2‐mM L‐glutamine and 1% (w/vol) fatty acid‐free BSA, and 180‐µL added/well. OCR and ECAR were measured ²¹ with some modifications.¹⁰ Six baseline rate measurements were made using a 2 min mix, 5 min measure cycle. Agonists (20 µL) were injected pneumatically by the machine into each well, mixed, and 10 measurements were made using the 2 min mix, 5 min measure cycle. OCR and ECAR rates immediately prior to compound injection were used as the basal rates and defined as 100%.

Dehvari N., Sato M., Bokhari M.H., Kalinovich A., Ham S., Gao J., Nguyen H.T., Whiting L., Mukaida S., Merlin J., Chia L.Y., Wootten D., Summers R.J., Evans B.A., Bengtsson T, & Hutchinson D.S. (2020). The metabolic effects of mirabegron are mediated primarily by β3‐adrenoceptors. Pharmacology Research & Perspectives, 8(5), e00643.

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Metabolic Flux Analysis of Astrocytes

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Primary astrocytes were cultured on Seahorse XF-24 or Seahorse XF-96 (Seahorse BioSciences, Billerica, MA, USA) plates at a density of 75,000 cells/well and 20,000 cells/well respectively. On the day of metabolic flux analysis, media was changed to Krebs-Henseleit buffer (KHB), pH 7.4, supplemented with 25 mM glucose and/or 1 mM pyruvate and incubated at 37°C in a non-CO₂ incubator for 1 h. All medium and injection reagents were adjusted to pH 7.4 on the day of assay. Using the Seahorse XF (Seahorse BioSciences) metabolic analyzer, 3 baseline measurements of oxygen consumption rate (OCR) were sampled prior to sequential injection of mitochondrial inhibitors. Three metabolic determinations were sampled following addition of each mitochondrial inhibitor prior to injection of the subsequent inhibitors. The mitochondrial inhibitors used were oligomycin (4 μM), FCCP (carbonyl cyanide 4-(trifluoromethoxy)- phenylhydrazone) (1 μM), and rotenone (1 μM). OCR was automatically calculated and recorded by the Seahorse software. After the assays, protein level was determined for each well to confirm equal cell density per well.

Jiang T, & Cadenas E. (2014). Astrocytic metabolic and inflammatory changes as a function of age. Aging Cell, 13(6), 1059-1067.

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