Wave software

Manufactured by Agilent Technologies

Sourced in United States, Germany

Wave software is a data analysis and visualization tool developed by Agilent Technologies. It is designed to provide users with the ability to acquire, analyze, and visualize data from various Agilent lab equipment and instrumentation.

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Seahorse Wave software v2.6 XFp software Wave 2.6 XF-96 Wave software Wave software 2.6.1 Seahorse Wave software Wave Software Version 2.4 XFe24 Wave software Wave analysis software Wave Controller Software Wave Desktop software

201 protocols using wave software

Evaluating Macrophage Oxygen Consumption

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For 72 h, ESdMs were incubated with LPS ± NaCl (40mM) to stimulate cells. Oxygen consumption rate (OCR) was determined under basal conditions and in response to 2 μM oligomycin, 1.5 μM FCCP, 100 nM rotenone plus 1 μM antimycin A (all Sigma-Aldrich). During OCR measurement activated ESdMs were cultured in XF-medium (XF Base Medium Minimal DMEM (Seahorse)) containing 10 mM glucose, 2 mM L-glutamin, and 1 mM sodium pyruvat (all from Sigma-Adlrich). OCR were evaluated using the Seahorse XFp Extracellular Flux Analyzer (Agilent Technologies) and analysed with Wave Software (Seahorse Bioscience).

Schmidt-Pogoda A., Strecker J.K., Liebmann M., Massoth C., Beuker C., Hansen U., König S., Albrecht S., Bock S., Breuer J., Sommer C., Schwab N., Wiendl H., Klotz L, & Minnerup J. (2018). Dietary salt promotes ischemic brain injury and is associated with parenchymal migrasome formation. PLoS ONE, 13(12), e0209871.

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Mitochondrial Function in Tumor Cells

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Mitochondrial function of tumor cells was measured using Mito stress test in XF96 extracellular flux analyzer (Seahorse Bioscience) according to the manufacturer’s instruction. Tumor cells (10,000 per well) were seeded in a 96-well Seahorse cell culture plate and incubated overnight. Next day, the cells were shifted to 1% O₂ condition (Hypoxia) for 6h at 37°C. After hypoxia treatment, the cells were washed twice and the media was replaced with DMEM sea horse media (Seahorse Bioscience) containing 1mM pyruvate (Sigma), 2mM glutamine (Gibco), and 10mM glucose (Sigma). Next, the plate containing the cells was kept in non-humidified 37°C incubator 1h prior to start of the experiment. Oxygen consumption rate (OCR) was measured at the basal level and after addition of the following compounds: oligomycin (Sigma, 0.5 μM), FCCP, (Sigma, 0.25 μM) and rotenone (Sigma, 0.5 μM). The data were analyzed using the wave software (Seahorse Bioscience) according to the manufacturer’s instructions. Proton leak was calculated by subtracting non-mitochondrial respiration from minimum rate measurement after oligomycin injection.

Pari A.A., Singhal M., Hübers C., Mogler C., Schieb B., Gampp A., Gengenbacher N., Reynolds L.E., Terhardt D., Géraud C., Utikal J., Thomas M., Goerdt S., Hodivala-Dilke K., Augustin H.G, & Felcht M. (2020). Tumor cell-derived Angiopoietin-2 promotes metastasis in melanoma. Cancer research, 80(12), 2586-2598.

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Analyzing Endothelial Cell Respiration

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An XFe24 extracellular flux analyser (Seahorse Biosciences, MA) was used to measure oxygen consumption rates [23 (link)]. Briefly, thirty thousand of treated endothelial cells were seeded in a provided 24-well plate in the CO₂ incubator. One hour before measurements, cells were removed and incubated at 37 °C in a normal atmosphere. The medium was then replaced with FX assay medium. Stock solutions of oligomycin (1 μM at final concentration), FCCP (0.5 μM at final concentration) and rotenone/antimycin A (0.5 μM/0.5 μM at final concentration) in an XF Cell Mito Stress Test Kit were prepared in FX assay medium and loaded into indicated injection ports respectively. Measurements were obtained at 37 °C. Assay cycles included 3 min of mixing, 2 min of waiting period, and 3 min of measurement. The Wave software provided by Seahorse Biosciences was used for data analysis. Basal respiration (Mean of Stage A – Mean of Non-mitochondria respiration (Stage D)), maximal respiration (Mean of Stage C – Mean of Non-mitochondria respiration (Stage D)) and ATP production (Mean of Stage A – (Mean of Non-mitochondria respiration+proton leak) (Stage B)) were determined by the XF Cell Mito Stress Test Generator that was provided by Seahorse Bioscience.

Xu W., Liang M., Zhang Y., Huang K, & Wang C. (2019). Endothelial FAM3A positively regulates post-ischaemic angiogenesis. EBioMedicine, 43, 32-42.

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Fatty Acid Oxidation Profiling

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Fatty acid oxidation was measured using Seahorse FAO assay, following the manufacturer’s protocol. Briefly, 1×10⁴ cells per well were seeded into a 96-well Seahorse assay plate with regular growth medium. After 24 hours, the medium was replaced with substrate-limited medium (0.5 mM glucose, 0.5 mM carnitine and 1% dialyzed FBS) supplemented with or without 1mM glutamine. After cultured for another 24 hours, the cells were washed twice with FAO assay medium (111 mM NaCl, 4.7 mM KCl, 1.25 mM CaCl2, 2 mM MgSO4, 1.2 mM NaH2PO4) supplemented with 2.5 mM glucose, 0.5mM carnitine and 5 mM HEPES, and then incubated in a non-CO₂ incubator for 45 minutes at 37°C. At the same time, the assay cartridge with cell mito stress compounds (final concentrations: 2 uM oligormycin, 1 uM CCCP, 2 uM antimycin A) were prepared and loaded onto the machine. 15 minutes prior to starting the assay, Etomoxir was added to the designated wells at the final concentration of 17 uM. After incubate for 15 minutes, BSA or palmitate-BAS (1 mM) was added to the designated wells and the plate was immediately loaded onto the machine to run the assay. The maximal respiration rate was analyzed using Seahorse Wave software.

Ni M., Solmonson A., Pan C., Yang C., Li D., Notzon A., Cai L., Guevara G., Zacharias L.G., Faubert B., Vu H.S., Jiang L., Ko B., Morales N.M., Pei J., Vale G., Rakheja D., Grishin N.V., McDonald J.G., Gotway G.K., McNutt M.C., Pascual J.M, & DeBerardinis R.J. (2019). Functional Assessment of Lipoyltransferase-1 Deficiency in Cells, Mice, and Humans. Cell reports, 27(5), 1376-1386.e6.

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Extracellular Acidification Rate Measurement

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The ECAR was evaluated using a Seahorse XF Glycolysis Stress Test Kit and measured by an XF96 Extracellular Flux Analyzer (Agilent, Santa Clara, USA) in accordance with the manufacturer's instructions as previously mentioned 34 . Briefly, 5-6×10³ cells were then plated into Seahorse XF96 cell culture microplates for 24 h. Before measurement, cells were incubated in a non-CO₂ injected incubator for 1 h to rule out its influence on the pH value, and baseline measurement was performed before the experiment started. Then, to ascertain the glycolytic flow and glycolytic capability of the investigated cells, glucose (10 mM), oligomycin (1 μM), an inhibitor of oxidative phosphorylation, and 2-deoxyglucose (2-DG; 50 mM) (both from Sigma Aldrich, USA) were utilized. Seahorse Biosciences Wave software was used to evaluate the data.

Luo Y., Pang B., Hao J., Li Q., Qiao P., Zhang C., Bai Y., Xiao C., Chen J., Zhi D., Liu Y., Dang E., Wang G, & Li B. (2023). Keratin 17 covalently binds to alpha-enolase and exacerbates proliferation of keratinocytes in psoriasis. International Journal of Biological Sciences, 19(11), 3395-3411.

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Extracellular Flux Analysis of Oxygen Consumption

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Oxygen consumption rate (OCR) was measured using an XF24 extracellular flux analyzer (Seahorse Bioscience, North Billerica, MA) in accordance with the manufacturer's protocol. In brief, cells were seeded in 24 well assay plates in culture medium and were incubated at 37°C in 5% CO₂ for 24 h. Then, the cells were changed to unbuffered RPMI 1640 (supplemented with 25 mmol/L glucose and 2 mmol/L L‐glutamine, without sodium bicarbonate and phenol red, Sigma) and incubated at 37°C in a non‐CO₂ incubator for 1 h. After four baseline measurements, indicated concentrations of drugs were injected and eight measurements were taken. Baseline normalized OCR at 1 h after the drug injection was calculated by Wave software (Seahorse Bioscience). 100% OCR was determined as the mean of OCR in the absence of drug. Data represent the mean of 3–4 independent wells.

Akatsuka A., Kojima N., Okamura M., Dan S, & Yamori T. (2016). A novel thiophene‐3‐carboxamide analog of annonaceous acetogenin exhibits antitumor activity via inhibition of mitochondrial complex I. Pharmacology Research & Perspectives, 4(4), e00246.

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Oxygen Consumption and Extracellular Acidification Rates

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Oxygen consumption and extracellular acidification rates were measured using an XFe96 Extracellular Flux Analyzer (Seahorse Bioscience) using the manufacturer’s established protocols. In brief, cells were plated overnight in Seahorse 96-well plates at 1 × 10⁴ per well in 80 μL of complete media. Cells were washed three times in non-buffered DMEM containing 25 mM glucose and 2 mM glutamine and incubated in this medium in a CO₂-free incubator at 37 °C for 2 h to allow for temperature and pH equilibration before loading into the XFe96 apparatus. In addition to basal measurements, the Mito Stress Test assay (Seahorse Bioscience, 103015-100) was performed according to manufacturer’s instructions. XFe assays consisted of sequential mix (3 min), pause (3 min), and measurement (5 min) cycles, allowing for determination of OCR/ECAR every 10 min. At the end of the assay media was removed from the plate which was then frozen at −80 °C for 24 h. CyQuant dye (Invitrogen, C7026) was then used according to the manufacturer’s instructions to generate DNA content values for data normalization. Data were analyzed using Wave software (Seahorse Bioscience).

Smith H.W., Hirukawa A., Sanguin-Gendreau V., Nandi I., Dufour C.R., Zuo D., Tandoc K., Leibovitch M., Singh S., Rennhack J.P., Swiatnicki M., Lavoie C., Papavasiliou V., Temps C., Carragher N.O., Unciti-Broceta A., Savage P., Basik M., van Hoef V., Larsson O., Cooper C.L., Vargas Calderon A.C., Beith J., Millar E., Selinger C., Giguère V., Park M., Harris L.N., Varadan V., Andrechek E.R., O’Toole S.A., Topisirovic I, & Muller W.J. (2019). An ErbB2/c-Src axis links bioenergetics with PRC2 translation to drive epigenetic reprogramming and mammary tumorigenesis. Nature Communications, 10, 2901.

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Extracellular Acidification in Macrophages

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Cellular extracellular acidification rate (ECAR) were measured in peritoneal macrophages. Measurements were taken using a Seahorse Bioscience XF96 extracellular analyzer. Three measurements were taken before and after addition of glucose (10 mM), oligomycin (1 μM), and 2-DG (50 mM). ECAR were calculated by the Seahorse Wave software.

Wang C., Chao Y., Xu W., Liu Z., Wang H, & Huang K. (2020). Myeloid FBW7 deficiency disrupts redox homeostasis and aggravates dietary-induced insulin resistance. Redox Biology, 37, 101688.

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Metabolic Profiling of Cells by Seahorse Assay

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Metabolic status was investigated on a Seahorse XF24 Analyzer (Agilent Technologies) with standard 24-well Seahorse microplates. A Mito Stress Test Kit (Agilent Technologies, #103015) was used to assess oxygen consumption ratio (OCR) and extracellular acidification rate (ECAR) after MC3324 treatment. In brief, 2 × 10⁴ cells were seeded into plates 12 h prior to analysis. The medium was then replaced with 175 μL of non-buffered RPMI containing 10 mM glucose, 2 mM glutamine, and 1 mM pyruvate. The cells were then treated with 25 μM MC3324 for 3 h at 37 °C and incubated in a CO₂-free incubator at 37 °C for 1 h to allow for temperature and pH equilibration before being loaded into the XF24 Analyzer. The injection sequence was programmed as follows: 1st, oligomycin (1 μM at final concentration); 2nd, carbonyl cyanide m-chlorophenylhydrazone (FCCP; 1 μM at final concentration); 3rd, rotenone and antimycin A (1 μM and 0.5 μM at final concentrations, respectively). Data were analyzed with Wave software (version 2.2.0, Seahorse Bioscience, Agilent Technologies, Santa Clara, CA, USA). Experiments were performed in triplicates. P-values were calculated using t-test. Statistical significance is expressed as ∗ p-value <0.05. Standard deviations are reported as error bars.

Chianese U., Papulino C., Passaro E., Evers T.M., Babaei M., Toraldo A., De Marchi T., Niméus E., Carafa V., Nicoletti M.M., Del Gaudio N., Iaccarino N., Randazzo A., Rotili D., Mai A., Cappabianca S., Mashaghi A., Ciardiello F., Altucci L, & Benedetti R. (2022). Histone lysine demethylase inhibition reprograms prostate cancer metabolism and mechanics. Molecular Metabolism, 64, 101561.

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Metabolic Profiling of A549 and H1299 Cells

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A549 or H1299 cells were seeded into an XF96 cell culture plate (Seahorse Bioscience) at a density of 1 × 10⁴ cells per well and incubated with complete medium overnight. ECAR and OCR were detected by a Seahorse XFe-96 Analyser (Agilent, Cheadle, Greater Manchester, UK). To monitor changes in ECAR, 10 mM glucose, 1.0 μM oligomycin and 50 mM 2-DG were used. Changes in OCR were assessed by sequential injection of final concentrations of 1.0 μM oligomycin, 0.5 μM FCCP and 0.5 μM rotenone and antimycin A. Corresponding metabolic parameters were further calculated by WAVE Software (Seahorse Bioscience, Agilent) and normalized based on the cell number of each well.

Liang L.J., Yang F.Y., Wang D., Zhang Y.F., Yu H., Wang Z., Sun B.B., Liu Y.T., Wang G.Z, & Zhou G.B. (2024). CIP2A induces PKM2 tetramer formation and oxidative phosphorylation in non-small cell lung cancer. Cell Discovery, 10, 13.

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