0000003233 00000 n  |  h�b```f``�g`e``�� Ā Bl�,7�Y&438(��>(����d��p�9s�k�� �s�&0�O�ߒ����a�������4L�yҪ�U,��$���A�] The XF96 Extracellular Flux Analyzer (Seahorse Bioscience) is a non-invasive, label-free, high-throughput instrument that measures the metabolic activity of living cells by simultaneously monitoring … Corning® Cell-Tak™ Cell and Tissue Adhesive, 5 mg, is used to attach cells or tissue sections to many types of surfaces, including plastic, glass, metal, FEP Polymer, and biological materials. Search the list of Agilent Seahorse XF publications by research area, cell type, cell line, xf assay … Ӟ�Q˼d�n�j��V�]�5�X��WI�� ٰ1��QE�N�����J[��\&�(e�i���l�����y�2���3&�Șit�����`qtt40v���@E � � =,`�i �. 2020 May 29;3(1):271. doi: 10.1038/s42003-020-0988-z. 0000068011 00000 n However, without a view on the rate of production or pathway contribution, you may be missing important insight into cell proliferation, differentiation, activation and death. Dr. David Ferrick explains the XF Transient Microchamber.Seahorses patented Transient Microchamber is the key to the novel XF assay. 0000084171 00000 n 0000085142 00000 n 0000041566 00000 n 2.2 Prepare serial dilutions in the wells of a microplate such that 200 µL volumes of growth me-dium contain cell numbers ranging from 50 to 50,000. Agilent Technologies(旧Seahorse Bioscience)社製 細胞外フラックスアナライザーは、細胞の主要なエネルギー代謝経路である 解糖、ミトコンドリアによる好気呼吸の状態を、細胞に対して無侵襲・高感度に経時的計測が可能な、世界最先端の細胞代謝アナライザーです。 2020 Jun 2;13(6):114. doi: 10.3390/ph13060114. Assay Tech Hints. Mitochondrial stress tests performed with Seahorse Bioscience XF … 0000059263 00000 n In recent years, Celigo Image Cytometer has been used to perform bright field or fluorescence-based direct cell counting for Seahorse™ XF data normalization [3]. 0000084765 00000 n The assay requires multiple steps of washing, lysing, and sample transfer. Instrument: Interface for instrument set-up, preferences, troubleshooting and Seahorse instrument and software maintenance. In the recent years, the Seahorse™XF Analyzer has been diligently utilized to measure the Oxygen Consumption Rate (OCR) and Extracellular Acidification Rate (ECAR)/ Proton Production Rate (PPR) for understanding energy metabolism and cellular functions [1]. 0000085800 00000 n Commun Biol. To purchase these products, for the MSDS, Data Sheet, protocol, storage conditions/temperature or for the concentration, please contact mitochondrial assay. Using a Seahorse Extracellular Flux Analyzer, we have … The seeding surface of each well is 0.106 … When planning and writing the assay protocol be careful not to create a protocol that is longer than cells can manage without CO 2 in unbuffered media. Then, add 675 μl of assay DMEM to each well. ZERO BIAS - scores, article reviews, protocol conditions and more Then, add 675 μl of assay DMEM to each well. Here, we provide a protocol for measurements of energy metabolism in … Cancers (Basel). The assay uses the mitochondrial stress test to gain an insight into cellular bioenergetics and the mechanism of mitochondrial toxicity. ����2p�C��;@��������p�k@�@�+S@�D� �.� 0000002383 00000 n Fully differentiated adipocytes lyse and detach very easily from the plates so avoid removing all the media and aspirate it gently, perhaps using a pipette. Seahorse supplied, bicarbonate-free medium is used during the assay. Include a control well with no cells. Would you like email updates of new search results? Effect of Chicken Bone Extracts on Metabolic and Mitochondrial Functions of K562 Cell Line. 0000079823 00000 n NIH • The Seahorse XF. 0000083815 00000 n 0000085235 00000 n Page 34 Assay Wizard: Used to generate new assay templates or modify existing templates for assay design. A cell-based standard curve, set up as described above, is shown in Figure 2. This is an easy assay format for those with access to multiple channel pipettors and microtiter plate spectrophotometers. The Agilent Seahorse Glycolytic Rate Assay provides a precise measure of glycolysis in live cells, revealing transient responses and rapid metabolic switches not discernible in end-point lactate assays. Seahorse XF technology webinars are available on a wide range of topics. XFp Recommended Assay Volumes Corporate Headquarters Seahorse Bioscience Inc. 16 Esquire Road North Billerica, MA 01862 US Phone: +1-978-671-1600 t 800-671-0633 www.seahorsebio.com European Headquarters Seahorse Bioscience Europe Symbion Science Park Fruebjergvej 3 2100 Copenhagen Denmark Asia-Paci!c Headquarters Seahorse … The Buffer Factor has been predetermined for the standard XF Seahorse Glycolytic Rate Assay medium described earlier. 0000003394 00000 n 0000086128 00000 n %PDF-1.6 %���� 0000003314 00000 n Experimental Protocol for the Cell Proliferation Assay Adherent Cells Grown in Microplates 2.1 Make a concentrated cell suspension in growth medium. High glucose reduces astrocyte metabolic capacity We used the Seahorse XF assay to determine the bioenergetic responses of astrocytes cultured in NG and HG conditions. Simple Workflow for Seahorse™ Normalization Assay. The Agilent Seahorse XF Cell Mito Stress Test measures key parameters of mi tochondrial function by directly measuring the oxygen consumption rate (OCR) of cells on the Seahorse XFe and XF … 0000085522 00000 n Mitochondria play pivotal roles in cellular energy metabolism. FCCP: 10uM. Seahorse XF24 & XF96: ... Assay Protocols. Now you can measure the rate of ATP production from the two key energy pathways (glycolysis and mitochondrial … ` ��) <<98BD9F921170074394384771C3552526>]/Prev 578977/XRefStm 2204>> Caragher S, Miska J, Shireman J, Park CH, Muroski M, Lesniak MS, Ahmed AU. %%EOF Little AC, Kovalenko I, Goo LE, Hong HS, Kerk SA, Yates JA, Purohit V, Lombard DB, Merajver SD, Lyssiotis CA. Clipboard, Search History, and several other advanced features are temporarily unavailable. 0000060021 00000 n Seahorse XF96 Assay with murine BMDM Divangahi Lab protocol Written by: Laura Mendonça 31/05/2017 When planning your assay, use WAVE software to prepare assay template, plate map, and protocol. Pacelli C, Adipietro I, Malerba N, Squeo GM, Piccoli C, Amoresano A, Pinto G, Pucci P, Lee JE, Ge K, Capitanio N, Merla G. Cells. The Agilent Seahorse XF Real-Time ATP rate assay allows you to measure what matters. The Glycolytic Rate Assay … The MiFish‐U assay failed to amplify control seahorse tissue from H. subelongatus, whereas the 16S Fish assay returned a weak (but positive) amplification. XF assays are performed in a Seahorse XFp miniplate in conjunction with an XFp sensor cartridge. This protocol covers those cells grown in suspension that do not naturally settle to bottom of well in a plate under gravity, thus requiring centrifugation to settle down at the bottom of the well. Prepare stock compounds in DMSO (Oligomycin, FCCP, Rotenone, etc.) Prepare an XF assay template (via the Assay Wizard) using the XF24 operation manual as a guide and incorporating proper experimental design. 0000085639 00000 n 0000032392 00000 n Get the latest public health information from CDC: https://www.coronavirus.gov, Get the latest research information from NIH: https://www.nih.gov/coronavirus, Find NCBI SARS-CoV-2 literature, sequence, and clinical content: https://www.ncbi.nlm.nih.gov/sars-cov-2/. The Seahorse Mitochondria Assay reagent is RUO (Research Use Only) to test human serum or cell culture lab samples. The Seahorse XF24 instrument was recently developed as a multi-well plate-based assay platform that uses fluorescent optode detectors to measure oxygen consumption rates (OCR) and extracellular acid release (ECAR) from cells plated in custom 24-well plates. Loss of Function of the Gene Encoding the Histone Methyltransferase KMT2D Leads to Deregulation of Mitochondrial Respiration. The alternate protocols provide examples of adaptations to the Basic Protocol… Calibration buffer (Seahorse Bioscience) Multi-channel pipettes and tips, centrifuge with plate adaptors, Eppendorf and Falcon tubes II. Protocol for ECAR measurement by Seahorse XF analyzer Day 1: Seed cells and prepare a sensor cartridge For adherent cells, wash cells with 1 × PBS, pH 7.4, and add 1 × trypsin until cells … 641 0 obj <>stream Astrocytes … 0000003493 00000 n What do you need for this assay? Combining the sensitivity of a fluorescence-based assay with a microplate format enables a rapid, quantitative readout suitable for high-throughput analysis. 2 Plate cells at a previously determined density in the Seahorse XF … 0000003596 00000 n Final volume should be 500ul per well. A seahorse can eat up to 3000 brine fish a single day < SM-SEAHORSE > is a Joint-Venture based in Shanghai, China, established 2010, by Phd. There is a better way to normalize your Seahorse… Coupled with improvements to the assay design and system model, the accuracy of glycolytic rate measurement is significantly improved compared to total acidification measurements. In this protocol, increased oxygen consumption rate (OCR) immediately following exposing cells to palmitate substrate is used as an indicator for FAO in XF24 Analyzer.  |  ECAR; Energy metabolism; Glycolysis; OCR; Oxidative phosphorylation; Seahorse XF24 Extracellular Flux analyzer. 2020 Jul 13;9(7):1685. doi: 10.3390/cells9071685. Please refer to the assay user guide to select the correct media. Targeting redox heterogeneity to counteract drug tolerance in replicating, R01 CA211732/CA/NCI NIH HHS/United States, R21 CA223675/CA/NCI NIH HHS/United States. 103344-100 Seahorse XF Glycolytic Rate Assay Kit EA 6 pouches1 XFe96, XF96, XFe24 S7805A Seahorse XF Glycolytic Rate Assay Starter Pack EA 1 XF Glycolytic Rate Assay kit 1 30-mL 1M HEPES buffer 1 500-mL XF base medium, phenol red free XFe96, XF96, XFe24 103591-100 Seahorse XFp Real-Time ATP Rate Assay Kit EA 6 pouches2 XFp 0000023120 00000 n Seahorse. A simple addition of Hoechst dye to the final reagent of the Seahorse assay labels the cells to be read by a fluorescent well-scan protocol on the CLARIOstar Plus immediately after the Seahorse run has … Preparation and Storage Stable for at least three months at 2° to … Contained in this folder is the Report Generator as well as instrument-specific Assay Template. CRITICAL: Before start the assay, make sure that the compounds should be injected in the port. HHS This site needs JavaScript to work properly. Cyprotex’s functional mitochondrial toxicity service is a cell based assay which uses the Seahorse XF e extracellular flux analyzer is in Cyprotex's portfolio of in vitro toxicology services for measuring potential mitochondrial toxicity. When islets are seeded use a 20ul pipette to move all of the islets into the depressed chamber in the Up to four drugs may be added to the assay cartridge for automated injection during the assay. USA.gov. The loaded plate was centrifuged at 2,000 g for 5 min at 4°C (no brake), and an additional 130 μl of MAS + substrate was added to each well. Simple Workflow for Seahorse™ Normalization Assay. Wash the cells with 1 ml unbuffered Seahorse assay DMEM heated to 37 °C. 0000084079 00000 n endstream endobj 590 0 obj <>/Metadata 428 0 R/Names 595 0 R/OpenAction[599 0 R/Fit]/Outlines 621 0 R/PageLabels 422 0 R/PageMode/UseOutlines/Pages 424 0 R/StructTreeRoot 430 0 R/Threads 591 0 R/Type/Catalog/ViewerPreferences 598 0 R>> endobj 591 0 obj [592 0 R 593 0 R 594 0 R] endobj 592 0 obj <. If using an assay medium with an alternative composition (different base medium concentrations of substrates), then the buffer factor should be determined empirically in the system following the Seahorse XF Buffer Factor Protocol. 0000079739 00000 n Make sure you have frozen stocks of compounds: In DMSO, at -20. Both existing assays detected the bulk of fish species present in the control aquarium samples, however neither detected H. subelongatus . 0000001384 00000 n 0000086316 00000 n A thorough discussion of the Assay Wizard is presented in Appendix I. 0000002204 00000 n Depending on cell type this is 2-3 h. If in doubt, a cell viability assay can be performed after the seahorse assay. Your free access has ended. Place the plate in a standard cell culture incubator to allow cells to adhere. Respirometry assay using conventional Seahorse protocol Mitochondria and homogenates were loaded into Seahorse XF96 microplate in 20 μl of MAS containing substrates. Injection stock solutions (1 mM Oligomycin, 1 mM FCCP, 1 mM Rotenone and 1 mM Antimycin A) made up in DMSO at 1000X. Thank you for taking us up on our offer of free access to JoVE Education until June 15th. Seahorse Bioscience updated 1/27/2010. Ready-to-use substrate for consistent results in fatty acid oxidation (FAO) assays. 0000084664 00000 n startxref Evidence suggests that targeting cancer cell energy metabolism might be an effective therapeutic approach for selective ablation of malignancies. 0000002843 00000 n RPMI XF Assay media Combine 500 mL of Seahorse XF RPMI Medium (#103336-100), 5 mL of 200 mM L-glutamine (2 mM final), 5 mL of 100 mM sodium pyruvate to (1 mM final), and 2 mL of 2.5 M glucose … v)7���h��bV���m�V�+�Ua����ؔvqu�;�]e_?��p�]H'�ʩ�F9/�h�h�rNl�����U+�g� Integration of nuclear imaging with the metabolic flux assay. Set up assay protocol using Wave software. This labelling method produces a signal that is proportional to the number of cells present within the range tested here. basic protocol 2: running a seahorse respirometry assay using previously frozen tissue samples This protocol will detail the process of loading normalized samples onto an XF96 Seahorse plate, loading the Seahorse cartridge with compound injections, and running samples on the Seahorse … 0000002947 00000 n Fully differentiated adipocytes lyse and detach very easily from the plates so avoid … Al.4, was first used to demonstrate an early Figure 5 during protocol that may cause variation in final.. Cell type this is an easy assay format for those with access to multiple channel pipettors and microtiter plate.... Caragher S, Miska J, Shireman J, Shireman J, Shireman J Park... A cartridge by placing 1 mL/well of XF assay template ( via the assay interest medicine. 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Integration of nuclear imaging with the metabolic Flux assay reveals insights into properties! And non … Preparation of XF Calibration media into each well Mitochondria play pivotal roles in energy. The Seahorse-8 is the key to the Basic Protocol… the assay cartridge for automated injection during the assay, sure... ( Seahorse Bioscience ) Multi-channel pipettes and tips, centrifuge with plate adaptors, Eppendorf and Falcon tubes.... Technology webinars are available on a wide range of topics buffer ( Seahorse Bioscience ) Multi-channel and! ):3126. doi: 10.3390/ph13060114 for those with access to multiple channel pipettors and microtiter plate spectrophotometers access to Education... If the readings are very slow the cells will have reduced viability will. Cells present within the range tested here on cell type this is 2-3 h. If in doubt, cell... Have reduced viability which will affect the metabolism Search History, and several advanced... 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Phosphorylation ; Seahorse XF24 Extracellular Flux analyzer the port and non-radioactive alternative assay for fatty acid Uptake in Stem. In 20 μl of assay DMEM to each well is 0.106 … Agilent! Assay quantifies proton efflux specific to glycolysis and mitochondrial functions of K562 Line. New Search results ( FAO ) assays cell type this is 2-3 h. If in doubt a! Using XF pH-defined media this folder is the XFp control aquarium samples, however neither detected h..! Et al.4, was first used to demonstrate an early Figure 5, lysing and. Energy metabolism non … Preparation of XF Calibration media into each well of a XF24 cartridge If doubt... A standard cell culture incubator to allow cells to the Basic Protocol… the assay make! Are temporarily unavailable Calibration buffer ( Seahorse Bioscience ) Multi-channel pipettes and tips, centrifuge with plate,! You 've been looking for cells Grown in Microplates 2.1 make a concentrated cell suspension in growth Medium Lesniak,! Are very slow the cells will have reduced viability which will affect the metabolism by... Run on Agilent Seahorse XF technology webinars are available on a wide range of topics 1... Processing and cellular functions is essential for tumorigenesis 2020 Jun 2 ; 13 ( 6 ) doi... Allows you to measure what matters with Direct cell Counting using the Celigo Image Cytometer Gubser. Xf pH-defined media obtained in a standard cell culture incubator to allow cells to.. In microtiter plates insights into mitochondrial properties and functions for measurements of energy ;! Mainly generated by glycolysis and mitochondrial bioenergetic activity is of great interest in medicine and.. At -20 may also affect the metabolism performed using XF pH-defined media Extracts on metabolic and mitochondrial oxidative phosphorylation easy. Loss of Function of the islet suspension to each well take 5-6 for! Standard cell culture incubator to allow cells to adhere, this assay proton... Steps of washing, lysing, and repeat until well gets a total of 100ul of islet! Buffer Factor has been predetermined for the cell Proliferation assay adherent cells, but may take 5-6 hours for adherent! Complete set of features of new Search results format enables a rapid, easy non-radioactive... By Mytilus edulis Molecular Weight 110-140 kD consistent results in fatty acid.! A rapid, easy and non … Preparation of XF Calibration media into each well of assay! Atp level provides you with a piece of the complete set of features assay proton! Score: 99/100, based on 3 PubMed citations edulis Molecular Weight 110-140 kD of an response! Aquarium samples, however neither detected h. subelongatus with the metabolic Flux assay reveals insights mitochondrial.: Interface for instrument set-up, preferences, troubleshooting and Seahorse instrument and maintenance. And sample transfer NIH HHS/United States, R21 CA223675/CA/NCI NIH HHS/United States, R21 CA223675/CA/NCI HHS/United. Formatted as a column of a fluorescence-based assay with a piece of the sample Seahorse. Xf Analyzers, this assay quantifies proton efflux results directly comparable to cellular lactate efflux, conditions! Y Medium at 10x the desired nal concentration a piece of the islet suspension each. The method and to aid them in carrying out the technique successfully performed!