Seahorse xf96 analyzer

Manufactured by Agilent Technologies

Sourced in United States, China, Belgium

The Seahorse XF96 analyzer is a laboratory instrument designed to measure the metabolic activity of cells in a high-throughput manner. It utilizes specialized microplates and sensors to quantify oxygen consumption rates and extracellular acidification rates, providing insights into cellular bioenergetics.

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Seahorse XF96 Extracellular Flux Analyzer (302 citations) XF96 analyzer (164 citations) Seahorse XF96 (83 citations) Seahorse XF96 Flux Analyzer (49 citations) Seahorse Analyzer (37 citations) Agilent Seahorse XF96 Extracellular Flux Analyzer (2 citations) Agilent Seahorse XF96 Analyzer (2 citations) Seahorse Bioscience XF96 analyzer (2 citations) Seahorse XF96 bioenergetic analyzer (2 citations) Seahorse XFe/XF96 Analyzer (2 citations)

215 protocols using seahorse xf96 analyzer

Seahorse XF96 Sensor Cartridge Preparation

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Timing: Day 2, 10 min

The Seahorse XF96 Sensor Cartridge and the Seahorse XF96 Cell Culture plate are put separately into the Seahorse XF96 Analyzer, with Seahorse XF96 Sensor Cartridge loaded first.

13.

Remove the hydrated Seahorse XF96 Sensor Cartridge from the 37°C incubators and put it into the Seahorse Analyzer.a.

Run the experiment that you have already designed with the Seahorse Wave Desktop Software on the Seahorse XF96 Analyzer. Once you click “run experiment”, the device will ask you to insert the hydrated sensor cartridge on top of the utility plate.

Remove the lid from the plate and put the hydrated Seahorse XF96 Sensor Cartridge into the device, with well A1 being the rear position on the left-back.

Confirm the insertion of the Seahorse XF96 Sensor Cartridge. The device will close and start equilibrating the plate automatically.

CRITICAL: Remove the lid from the Seahorse XF96 Sensor Cartridge before putting it into the device. Not removing the lid will not give you results and might damage the device.

Burgstaller S., Bischof H., Lukowski R., Graier W.F, & Malli R. (2021). Investigating the K+ sensitivity of cellular metabolism by extracellular flux analysis. STAR Protocols, 2(4), 100876.

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Metabolic Analysis of RAW 264.7 Cells

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Glucose uptake and lactate secretion were determined by measuring the concentration of glucose and lactate in cell culture supernatants and control media in a YSI 2950D Biochemistry Analyzer, and quantification was achieved by measuring respective standards. OCR was determined using a Seahorse XF-96 Analyzer (Agilent). Briefly, 2 × 10⁴ RAW 264.7 cells were plated and incubated overnight on Seahorse XF-96 Cell Culture Microplates (Agilent). On the second day, cell culture media were changed to Seahorse XF RPMI media (Agilent) containing 10 mM glucose, 2 mM glutamine and 10 mM itaconate or mesaconate, and then cells were incubated for 4 h. After loading to the Seahorse XF-96 Analyzer, a sequential in situ incubation of components was performed as follows: 10 ng ml⁻¹ LPS for 3 h, 1.5 μM oligomycin (Agilent) for 18 min, 1 μM FCCP (Agilent) for 18 min and 0.5 μM rotenone plus 0.5 μM antimycin A (Agilent) for 18 min. OCR data were analysed with Wave software (Agilent).

He W., Henne A., Lauterbach M., Geißmar E., Nikolka F., Kho C., Heinz A., Dostert C., Grusdat M., Cordes T., Härm J., Goldmann O., Ewen A., Verschueren C., Blay-Cadanet J., Geffers R., Garritsen H., Kneiling M., Holm C.K., Metallo C.M., Medina E., Abdullah Z., Latz E., Brenner D, & Hiller K. (2022). Mesaconate is synthesized from itaconate and exerts immunomodulatory effects in macrophages. Nature metabolism, 4(5), 524-533.

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Mitochondrial Respiration Measurement in THP1 Cells

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Oxygen consumption rate was determined using a Seahorse XF-96 Analyzer (Agilent) and the Mito Stress Test kit (Agilent, catalogue no. 103015-100) following the manufacturer’s protocols. Briefly, 2.5 × 10⁴ THP1 cells were plated on Agilent Seahorse XF96 cell culture microplates (part no. 101085-004) and differentiated with phorbol 12-myristate 13-acetate as described above. On the day of the assay, the cell culture medium was changed to Seahorse XF RPMI medium (Agilent, catalogue no. 103576-100) containing 10 mM glucose (Agilent, catalogue no. 103577-100), 1 mM pyruvate (Agilent, catalogue no. 103578-100) and 2 mM glutamine (Gibco, catalogue no. 35050-038), and placed in a 37 °C non-CO₂ incubator for 45–60 min before the assay. Upon loading to the Seahorse XF-96 Analyzer, sequential in situ incubation of components was performed as follows: baseline measurement for 18 min, 1.5 µM oligomycin (Agilent) for 18 min, 1 µM carbonyl cyanide-p-trifluoromethoxyphenylhydrazone (Agilent) for 18 min and 0.5 µM rotenone/antimycin A (Agilent) for 18 min. Oxygen consumption rate data were analysed with Wave v.2.2.1 software (Agilent).

Chen F., Elgaher W.A., Winterhoff M., Büssow K., Waqas F.H., Graner E., Pires-Afonso Y., Casares Perez L., de la Vega L., Sahini N., Czichon L., Zobl W., Zillinger T., Shehata M., Pleschka S., Bähre H., Falk C., Michelucci A., Schuchardt S., Blankenfeldt W., Hirsch A.K, & Pessler F. (2022). Citraconate inhibits ACOD1 (IRG1) catalysis, reduces interferon responses and oxidative stress, and modulates inflammation and cell metabolism. Nature Metabolism, 4(5), 534-546.

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Mitochondrial Respiration Profiling

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Oxygen consumption rate (OCR) was measured using Seahorse XF96 Analyzers (Agilent, Santa Clara, CA, USA) according to the manufacturer’s protocol. Prior to the start of the experiment, cells were seeded (8000 cells/well) into the XF96 cell culture plate and allowed to adhere for 24 h. Cell culture media were replaced with XF media (Seahorse Bioscience, North Billerica, MA, USA) supplemented with 2 mmol/L Glutamax, 1 mmol/L sodium pyruvate, and 25 mmol/L glucose and were incubated for 1 h in a 37 °C CO₂ incubator. Following the establishment of a basal OCR reading, the following pharmacological manipulators of mitochondrial respiratory chain proteins were injected sequentially: (i) oligomycin (1 μmol/L) at ATP synthase inhibitor; (ii) carbonyl cyanide-4-(trifluoromethoxy) phenyl hydrazone (FCCP) (1.5 μmol/L), as a mitochondrial uncoupler; followed by (iii) antimycin A (10 μmol/L), as a complex III inhibitor. The results were calculated using the Seahorse XF Mito Stress Test Report Generator (Seahorse Bioscience, North Billerica, MA, USA).

Cao X., Wu Y., Hong H, & Tian X.Y. (2022). Sirtuin 3 Dependent and Independent Effects of NAD+ to Suppress Vascular Inflammation and Improve Endothelial Function in Mice. Antioxidants, 11(4), 706.

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Seahorse XF Mitochondrial Respiration Assay

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Assays were performed using a Seahorse XF 96 analyzers (Agilent, USA) according to the manufacturer’s instructions. Concisely, 8 × 10³ cells/well were seeded in a 96-well XF cell culture microplate and incubated overnight in the recommended growth medium before the assay. The sensor cartridge was hydrated in a Seahorse XF Calibrant at 37 °C in a CO₂-free incubator overnight. Subsequently, the medium was replaced with CO₂-free low-buffered assay medium containing 2 mM L-glutamine (Lonza, 17-605E), 1 mM pyruvate (Sigma, S8636) and 25 mM glucose (Sigma, G7528). The oxygen-consumption rate (OCR) was measured at baseline and following sequential addition of 2 µM oligomycin (Abcam, ab141829), 1 µM FCCP (Sigma, C2920), 5 µM rotenone (Sigma, C2920), and 5 µM antimycin A (Abcam, ab141904). We used the total number of cells per well to normalize the OCR data.

Zhao H., Cheng X., Yan L., Mi F., Wang W., Hu Y., Liu X., Fan Y., Min Q., Wang Y., Zhang W., Wu Q, & Zhan Q. (2024). APC/C-regulated CPT1C promotes tumor progression by upregulating the energy supply and accelerating the G1/S transition. Cell Communication and Signaling : CCS, 22, 283.

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Metabolic Assessment of HCMEC/D3 Cells

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Pretreated HCMEC/D3s were seeded in Seahorse XF96 plates (Agilent, China) with the amount of 20,000 cells per well. Cells were incubated with nonglucose medium for 1 h before undergoing the glycolysis stress evaluation (Seahorse XF96 analyzers, Agilent, China) by detecting the extracellular acidification rate (ECAR) of cells. Detection was carried out per 5 min before and after sequentially adding with glucose, oligomycin and 2‐dexoy‐d‐glucose (2‐DG) (Agilent, China). Next, cells were incubated with normal medium for 1 h before the mitochondrial stress evaluation by detecting the oxygen consumption rate (OCR) of cells. And detection was carried out per 5 min before and after sequentially adding with oligomycin, carbonyl cyanide 4‐(trifluoromethoxy)phenylhydrazone (FCCP) and rotenone/antimycin A (Agilent, China). Wave software and Graphpad were employed to process and analyze the data obtained.²⁵

Shu J., Wang C., Tao Y., Wang S., Cheng F., Zhang Y., Shi K., Xia K., Wang R., Wang J., Yu C., Chen J., Huang X., Xu H., Zhou X., Wu H., Liang C., Chen Q., Yan S, & Li F. (2023). Thermosensitive hydrogel‐based GPR124 delivery strategy for rebuilding blood‐spinal cord barrier. Bioengineering & Translational Medicine, 8(5), e10561.

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Seahorse XF96 Analysis of Cellular Respiration

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Seahorse XF96 analyzers (Seahorse Biosciences, North Billerica, MA, USA) were used to assess the cellular oxygen consumption rate (OCR) and extracellular acidification rate (ECAR). Briefly, HUVECs were seeded in XF-96 cell culture plates (Seahorse Bioscience) at 1*10⁴ cells/well and incubated under standard conditions for 24 h. Cells were washed with XF Base RPMI (Seahorse Bioscience #103336, North Billerica, MA, USA) containing 8 mM glucose, 8 mM pyruvate, and 2 mM L-glutamine. The overall oxygen consumption rate was measured during the addition of GYY4137 (0.1 mM, 1 mM,10 mM)( Sigma-Aldrich, #SML0100, St. Louis, MI, USA) and ZnCl₂ (Sigma-Aldrich #3208086, St. Louis, MI, USA). Experiments were conducted using six replicates for each condition and repeated in two independent experiments. Data were analysed by using Wave Desktop and Controller 2.6 Software.

Star B.S., van der Slikke E.C., Ransy C., Schmitt A., Henning R.H., Bouillaud F, & Bouma H.R. (2023). GYY4137-Derived Hydrogen Sulfide Donates Electrons to the Mitochondrial Electron Transport Chain via Sulfide: Quinone Oxidoreductase in Endothelial Cells. Antioxidants, 12(3), 587.

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Mitochondrial Respiration and Glycolysis Assay

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Assays were performed using the Seahorse XF 96 analyzers (Seahorse Bioscience, Agilent). OCR measurements were performed by using Agilent Seahorse XF Mito Stress Test Kit (103015-100) according to the manufacturer’s instruction. Briefly, 8 × 10³ cells/well were seeded in a 96-well XF cell culture microplate in growth medium 24 h before assay. Hydrate a sensor cartridge in a Seahorse XF Calibrant at 37 °C in a non-CO₂ incubator overnight. And then OCR was measured with an XF96 analyzer in XF base medium (pH 7.4) containing 1 mM pyruvate, 2 mM glutamine, and 10 mM glucose following sequential addition of Oligomycin (4 μM), FCCP (1 μM) and antimycin A (0.5 μM). Data were analyzed by the Seahorse XF Mito Stress Test Reporter Generator package. ECAR measurements were performed by using Agilent Seahorse XF Mito Stress Test Kit (103020-100) according to the manufacturer’s instruction. The process of ECAR measurements was almost the same with OCR measurements, except the sequential addition of Glucose (10 mM), Oligomycin (1 μM), 2-DG (50 mM).

Gong W., Xu J., Wang Y., Min Q., Chen X., Zhang W., Chen J, & Zhan Q. (2022). Nuclear genome-derived circular RNA circPUM1 localizes in mitochondria and regulates oxidative phosphorylation in esophageal squamous cell carcinoma. Signal Transduction and Targeted Therapy, 7, 40.

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Mitochondrial Respiration in PBMCs

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Whole blood was collected into EDTA tubes by venous puncture for all participants. Buffy coats were submitted to a Ficoll-Paque Plus (GE Healthcare) gradient centrifugation for isolation of the mononuclear fraction. PBMCs were cryopreserved in Fetal Bovine Serum with 10% DMSO. Cells were thawed within 6 weeks of isolation and underwent assessment using the Seahorse XF96 Analyzer (Seahorse Bioscience) to quantify oxygen consumption rate (OCR) using the mitochondrial stress test assay. Specifically, PBMCs were plated at 500,000 cells/well and 3 OCR measurements were taken sequentially on 5-6 technical replicate wells prior to and upon serial injection of 3.5 μM oligomycin (Sigma; complex V inhibitor), 1 μM fluoro-carbonyl cyanide phenylhydrazone (FCCP; Sigma; mitochondrial oxidative phosphorylation uncoupler) and 14 μM rotenone + 14 μM antimycin A (Sigma; complex I and III inhibitors, respectively) to evaluate measures of mitochondrial respiration including basal respiration, ATP-linked respiration, proton leak, maximal respiration, spare respiratory capacity (SRC) and non-mitochondrial respiration. All bioenergetic data were normalized to protein in the well for subsequent analyses. For data calculation, the Seahorse Wave software (v2.2.0) was used.

Donald P.R., Sirgel F.A., Kanyok T.P., Danziger L.H., Venter A., Botha F.J., Parkin D.P., Seifart H.I., Van de Wal B.W., Maritz J.S, & Mitchison D.A. (2000). Early Bactericidal Activity of Paromomycin (Aminosidine) in Patients with Smear-Positive Pulmonary Tuberculosis. Antimicrobial Agents and Chemotherapy, 44(12), 3285-3287.

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Mitochondrial Respiration Profiling Using Seahorse

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The oxygen consumption rate (OCR), an indicator of mitochondrial respiration, was measured using the Seahorse XF96 Analyzer (Seahorse Bioscience, USA). Treated and untreated cells were seeded in XF 96-well plates and incubated overnight at 37°C under 5% CO2 humidified atmosphere. Initially, the cells were incubated in the normal medium, and OCR was assessed. Following this, oligomycin (2.0 μM), carbonyl cyanide-p-trifluoromethoxyphenylhydrazone (FCCP) (0.3 μM), and antimycin A and rotenone (0.5 μM) were injected in turn, and OCRs were assessed. The key parameters of mitochondrial function, coupling efficiency and spare respiratory capacity, were calculated by calculated by the XF Cell Mito Stress Test software.

Han D., Wei W., Chen X., Zhang Y., Wang Y., Zhang J., Wang X., Yu T., Hu Q., Liu N, & You Y. (2015). NF-κB/RelA-PKM2 mediates inhibition of glycolysis by fenofibrate in glioblastoma cells. Oncotarget, 6(28), 26119-26128.

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