Seahorse xf96 cell culture plate

Manufactured by Agilent Technologies

Sourced in United States

The Seahorse XF96 cell culture plate is a specialized microplate designed for use with the Seahorse XF Analyzer. It enables the real-time measurement of cellular metabolism, including oxygen consumption rate (OCR) and extracellular acidification rate (ECAR), in a 96-well format.

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13 protocols using seahorse xf96 cell culture plate

Seahorse XF96 Cell Assay Timing and Handling

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Timing: Day 2, variable, depending on the protocol

While preparing the cells for the assay and after loading the Seahorse XF96 Sensor Cartridge, the device will ask you for the Seahorse XF96 Cell Culture plate. Load the Seahorse XF96 Cell Culture plate when all incubation steps have been finished.

15.

Run the assay.a.

Once the device asks you for the Seahorse XF96 Cell Culture plate, insert the plate after incubation times are completed and confirm the insertion of the plate to start the experiment.

During the experiment, the device will show you the measured values. Troubleshooting 2, 3, 4, and 5.

CRITICAL: The Incubation times of cells should not be shortened. Put the plate into the device immediately after the respective incubation times. Avoid spill over from the different wells during repetitive handling of the plates.

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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Extracellular Acidification Rate Analysis

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The Seahorse XF 96 Extracellular Flux Analyzer (Agilent) was used to determine the extracellular acidification rate (ECAR). According to the manufacturer’s instructions, ECAR was examined with a Seahorse XF glycolysis stress test kit. Briefly, 2 × 10⁴ HCT116 or SW480 cells per well with different treatments were seeded into a Seahorse XF 96 cell culture plate with 15% fetal bovine serum DMEM overnight. Cells were washed and incubated with base medium with 2 mML-glutamine for 1 h at 37 °C, CO2-free incubator. After 3 baseline measurements, glucose, oligomycin, and 2-DG was sequentially added d into each well at the time points specified to a final concentration of 10 mM, 10 μM or 50 mM, respectively. ECAR dates were assessed by Seahorse XF 96 Wave software.

Sun Z., Zhang Q., Yuan W., Li X., Chen C., Guo Y., Shao B., Dang Q., Zhou Q., Wang Q., Wang G., Liu J, & Kan Q. (2020). MiR-103a-3p promotes tumour glycolysis in colorectal cancer via hippo/YAP1/HIF1A axis. Journal of Experimental & Clinical Cancer Research : CR, 39, 250.

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Glycolytic Profiling of Cultured Cells

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For lactate production analysis, the LA concentration in the supernatants of cells cultured for 24 hours was measured enzymatically using a Lactate-Glo Assay from Promega (#J5021, Madison, Wisconsin, USA). For glucose-uptake production analysis, the cells were cultured for 24 hours with an associated fresh medium before harvesting these cells. Intracellular glucose concentration was quantified enzymatically using a Glucose Uptake-Glo Assay from Promega (#J1341, Madison, Wisconsin, USA, USA). For the Seahorse glycolysis analyzer: 10 000 cells/well were seeded in a Seahorse XF96 cell culture plate and were allowed to adhere overnight. The next day, plates were further incubated at 37°C in a non-CO2 incubator for 30 min, followed by testing with Glycolytic Rate Assay Kit of Agilent Seahorse XF (#103344-100). One hour before measurement, cell culture media was replaced with Seahorse XF DMEM with pH 7.4, containing 1 mM pyruvate, 2 mM glutamine, and 10 mM glucose. The following concentrations of each drug were used for extracellular acidification rate (ECAR) acquisitions: 2-DG 50 µM, Rotenone and Antimycin A, 0.5 µM. All reagents were from Agilent (Santa Clara, California, USA).

Ren Y., Kumar A., Das J.K., Peng H.Y., Wang L., Balllard D., Xiong X., Ren X., Zhang Y., Yang J.M, & Song J. (2022). Tumorous expression of NAC1 restrains antitumor immunity through the LDHA-mediated immune evasion. Journal for Immunotherapy of Cancer, 10(9), e004856.

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Extracellular Acidification Profiling

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The Seahorse XF 96 Extracellular Flux Analyzer (Agilent) was used to determine the extracellular acidi cation rate (ECAR). According to the manufacturer's instructions, ECAR was examined with a Seahorse XF glycolysis stress test kit. Brie y, 2×10 4 HCT116 or SW480 cells per well with different treatments were seeded into a Seahorse XF 96 cell culture plate with 15% fetal bovine serum DMEM overnight. Cells were washed and incubated with base medium with 2 mML-glutamine for 1 h at 37 °C, CO2-free incubator. After 3 baseline measurements, glucose, oligomycin, and 2-DG was sequentially added d into each well at the time points speci ed to a nal concentration of 10 mM, 10 μM or 50 mM, respectively. ECAR dates were assessed by Seahorse XF 96 Wave software.

Sun Z., Zhang Q., Yuan W., Li X., Chen C., Guo Y., Shao B., Dang Q., Zhou Q., Wang Q., Wang S., Liu J., & Kan Q. (2020). MiR-103a-3p promotes tumour glycolysis in colorectal cancer via Hippo/YAP1/HIF1A axis.

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Mitochondrial Respiration Analysis in HAP1 Cells

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All the respiration measurements were performed using Seahorse XFe96 Analyzer (Agilent). The HAP1 cells were seeded in Seahorse XF96 cell culture plate (Agilent) at a density of 3 × 10⁴ to 3.3 × 10⁴ cells per well overnight. On the subsequent day, cells were washed and incubated in basic DMEM media (D5030; Sigma-Aldrich) supplemented with glucose, glutamine, and pyruvate at 37°C in non-CO₂ incubator 1 h before the assay. Mitochondrial respiration function was measured using Seahorse XF Cell Mito Stress Test kit (Agilent) according to the manufacturer’s instructions. Briefly, the delivery chambers of the sensor cartridge were loaded with Oligomycin (F₁F_o–ATPase synthase inhibitor) or FCCP (uncoupler) or Rotenone and Antimycin (Complex I and Complex III inhibitor, respectively) to measure basal, proton leak, maximal, and residual respiration in XFe96 Analyzer. Cell number was normalized after the run using Hoechst staining. Data were analyzed using wave software (Agilent).

Anand R., Kondadi A.K., Meisterknecht J., Golombek M., Nortmann O., Riedel J., Peifer-Weiß L., Brocke-Ahmadinejad N., Schlütermann D., Stork B., Eichmann T.O., Wittig I, & Reichert A.S. (2020). MIC26 and MIC27 cooperate to regulate cardiolipin levels and the landscape of OXPHOS complexes. Life Science Alliance, 3(10), e202000711.

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Mitochondrial Function and Glycolytic Capacity

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Seahorse XFe96 Extracellular Flux Analyser (Agilent Technologies, Billerica, MA, USA) was used to detect glycolytic capacity and cellular mitochondrial function. Measurements were performed using Seahorse Glycolysis and XF cell Mito Stress Test kits (Seahorse Bioscience, Billerica, MA, USA). Cells were seeded in an Agilent seahorse XF96 cell culture plate at a density of 4 × 10⁴ cells per well and cultured with prepared XF medium (seahorse, 102353-100) the day before determination. The base medium, DMEM, was supplemented with 10 mM glucose, 2 mM glutamine and 2 mM pyruvate. The cell plate was placed in a non-CO₂ incubator for 1 h prior to the assay. After monitoring baseline respiration, for ECAR measurement, 10 mM glucose, 2 μM oligomycin and 50 μM 2-DG were automatically injected into each well. In order, 2 μM oligomycin, 1 μM FCCP and 0.5 μM rotenone/antimycin were used to measure OCR. OCR and ECAR values were analyzed using Wave 2.6 software (Agilent Technologies) after the number of cells was renormalized.

Jin Y., Bian S., Wang H., Mo J., Fei H., Li L., Chen T, & Jiang H. (2022). CRMP2 derived from cancer associated fibroblasts facilitates progression of ovarian cancer via HIF-1α-glycolysis signaling pathway. Cell Death & Disease, 13(8), 675.

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Mitochondrial Respiration Profiling of NME1-treated SH-SY5Y Cells

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SH-SY5Y cells were plated in a V3-PS TC-treated Agilent Seahorse XF96 cell culture plate at a density of 3 × 10⁴ cells per well. It was ensured that SH-SY5Y cells were at a passage number lower than 30 for each run. At 24 h after plating, the cells were treated with 100 ng/ml of NME1 for a duration of 48 h. The oxygen consumption rate (OCR) was measured using a Cell Mito Stress Test Kit (Cat No. 103015-100) from Agilent according to the manufacturer’s instructions using a MitoXF96 analyzer. The kit focuses on several aspects of cellular respiration using oligomycin (ATP-synthase inhibitor), FCCP (Protonophore, uncouples mitochondrial oxidative phosphorylation) and rotenone (inhibits mitochondrial complex I, thereby inhibiting mitochondrial electron transport chain). The OCR values were used to calculate basal respiration, proton leak, maximal respiration and ATP production rate. The cells were lysed in RIPA buffer after the conclusion of the assay and the protein levels were measured using bicinchoninic acid (BCA) assay ThermoFisher Scientific (Cat No. 23227) to ensure uniform protein content across groups.

Anantha J., Goulding S.R., Tuboly E., O’Mahony A.G., Moloney G.M., Lomansey G., McCarthy C.M., Collins L.M., Sullivan A.M, & O’Keeffe G.W. (2021). NME1 Protects Against Neurotoxin-, α-Synuclein- and LRRK2-Induced Neurite Degeneration in Cell Models of Parkinson’s Disease. Molecular Neurobiology, 59(1), 61-76.

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Measuring Mitochondrial Respiration in HAP1 Cells

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All respiration measurements were performed using Seahorse XFe96 Analyzer (Agilent). The HAP1 cells were seeded into Seahorse XF96 cell culture plate (Agilent) at a density of 30,000 cells per well overnight. Next day, cells were washed and incubated in basic DMEM media (Sigma, D5030) supplemented with glucose, glutamine and pyruvate at 37°C in non‐CO₂ incubator 1 h prior to the assay. Mitochondrial respiration function was measured using Seahorse XF Cell Mito Stress Test Kit (Agilent) according to the manufacturer's instructions. Briefly, the delivery chambers of the sensor cartridge were loaded with oligomycin (F₁F_O‐ATPase synthase inhibitor) or FCCP (uncoupler) or rotenone and antimycin (complex I and complex III inhibitors, respectively) to measure basal, proton leak, maximum and residual respiration. Cell number was normalized after the run using Hoechst staining. Data were analysed using wave software (Agilent).

Kondadi A.K., Anand R., Hänsch S., Urbach J., Zobel T., Wolf D.M., Segawa M., Liesa M., Shirihai O.S., Weidtkamp‐Peters S, & Reichert A.S. (2020). Cristae undergo continuous cycles of membrane remodelling in a MICOS‐dependent manner. EMBO Reports, 21(3), e49776.

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Measuring Hepatocyte Mitochondrial Function

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Isolated hepatocytes (12,000 per well) were seeded into a Seahorse XF96 cell culture plate (Agilent Technologies) after having been coated with collagen. Cells were kept in recovery media for 4 – 6 h and then washed with DMEM media (low glucose plus 10% FBS) and incubated overnight (DMEM low glucose (5 mM) with 10% FBS, 1 nM insulin, 1 nM dexamethasone, and antibiotics (10,000 units/mL penicillin and 10 mg/mL streptomycin, Invitrogen). The following morning, cells were washed and incubated in 180 μL of prewarmed (~37°C) DMEM base media (D5030, Sigma) supplemented with 2mM L-glutamine, 24mM bicarbonate, 10mM HEPES and 0.2% BSA with or without PKa 10μM and equilibrated in a ~37°C non CO2 incubator for 1 h prior to the study. Basal oxygen consumption was observed followed by the addition of the gluconeogenic substrate combination of pyruvate (1 mM) and lactate (9 mM). Chemical stimulants for assessing mitochondrial function were subsequently injected following the substrate addition in the following order; oligomycin 5 μM, FCCP 10 μM and rotenone 5 μM. Measurements of oxygen consumption rate were taken, and the average of the measurements after each injection was used for analysis.

Abulizi A., Cardone R.L., Stark R., Lewandowski S.L., Zhao X., Hillion J., Ma L., Sehgal R., Alves T.C., Thomas C., Kung C., Wang B., Siebel S., Andrews Z.B., Mason G.F., Rinehart J., Merrins M.J, & Kibbey R.G. (2020). Multi-Tissue Acceleration of the Mitochondrial Phosphoenolpyruvate Cycle Improves Whole-Body Metabolic Health. Cell metabolism, 32(5), 751-766.e11.

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Analyzing Mitochondrial ATP Dynamics in BMDMs

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BMDMs were seeded on a Seahorse XF96 cell culture plate (Agilent, #102601–100) overnight in DMEM containing 10% FBS. The following day BMDMs were treated with vehicle or 1 nM RvD1 20 mins prior to addition of NCs at a ratio of 1:2 as above for 2 hrs (37°C, 5% CO₂₎. To remove non-ingested dead cells, BMDMs were washed with the Seahorse XF DMEM media pH = 7.4 (Agilent, #103575–100) supplemented with 1 mM pyruvate (Sigma, Cat #S8638), 10 mM glucose (Sigma, Cat #G8769), 2 mM glutamine (Sigma, Cat #G8540) and then incubated at 37°C without CO₂ for 1 hr. For measuring mitochondrial ATP production in live cells, oxygen consumption rate (OCR) was measured using the Seahorse XFe Real-Time ATP Rate assay kit (Agilent, #103592–100) and the Seahorse XF96 Extracellular Flux Analyzer (Agilent Technologies, Santa Clara, CA). After three basal OCR measurements, the mitochondrial inhibitors oligomycin (1.5 μM) and Rotenone/Antimycin A (0.5 μM) were serially injected after every three measurements. The Mito ATP production rate was calculated according to equations described in the Agilent Seahorse XF Real-Time ATP Rate Assay user guide.

Hosseini Z., Marinello M., Decker C., Sansbury B.E., Sadhu S., Gerlach B.D., Ramos R.B., Adam A.P., Spite M, & Fredman G. (2021). Resolvin D1 enhances necroptotic cell clearance through promoting macrophage fatty acid oxidation and oxidative phosphorylation. Arteriosclerosis, thrombosis, and vascular biology, 41(3), 1062-1075.

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