Sodium bicarbonate-free Ham’s F12 (21700-075, Life Technologies, Inc.); store at 4 °C. Seahorse™ Normalization Assay at a Glance Simple Workflow for Seahorse™ Normalization Assay In recent years, Celigo Image Cytometer has been used to perform bright field or fluorescence-based … The primordium of the brood pouch first appears as linear projections at both ventrolateral sides of the body. Isoproterenol (12760, Sigma–Aldrich Corp.). Its unique highly ordered lamination, trisynaptic circuitry, and site for neurogenesis has contributed a wealth of information toward our current understanding of experience-dependent neuroplasticity at the systems, cellular, and molecular levels. OCR is reported in units of pmol/minute and ECAR in mpH/minute. They eat for the most of the time, as they … Seahorse consumables are developed specifically for use with the Seahorse XF Analyzer. New resources are continually being developed to ensure you have the tools you need to be successful in your research area. 4 (cited from Wilson et al., 2003). The pellet is then resuspended in the 13-C-containing labeling medium and cultured at 37 °C for several hours depending the 13-C substrate and on which metabolic flux is under investigation. Seahorse XFp Extracellular Flux Analyzer The role of metabolism in cellular and physiological processes from cardiovascular function to immune response is well established, with many diseases now linked … Interestingly, based on recent molecular studies, genes involved in these functions of brood pouch are commonly expressed in female internally-gestating fish and also in viviparous amniotes (Whittington et al., 2015). Wash the cells with 1 ml unbuffered Seahorse assay DMEM heated to 37 °C. 2 mM Glutamax II (Gibco #35050-061, Life Technologies, Inc.); store at − 20 °C. However, the Seahorse approach could reveal if a given cell type is able or not able to oxidize sulfide and would allow at least semiquantitative studies as the fastest rate observed is likely to approach the maximal rate of oxygen consumption possible in the presence of sulfide. The treatment of data would need to consider the evolution of oxygen concentration in individual wells (Fig. According to Linton and Soloff (1964), this is not essential because embryos can develop even during in vitro incubation. They provide sensitive, kinetic measurements to simultaneously analyze the two main metabolic energy pathways: mitochondrial respiration and glycolysis. Seahorse Food: The most interesting thing about seahorses is the astonishing fact that they don’t have a stomach. BMP7 (Pepro Tech Inc.); store at − 20 °C. Mathias Wenes, ... Lianjun Zhang, in Methods in Enzymology, 2020. OCR and ECAR rates are key indicators of mitochondrial respiration and glycolysis and these measurements provide a systems-level view of cellular metabolic function in cultured cells and ex-vivo samples. Your access has now expired. The T cells are first collected, centrifuged and washed once in RPMI medium without d-glucose and l-glutamine. Designing these protocols and detecting the metabolites by mass spectrometry requires expert knowledge and is thus generally performed in collaboration with metabolomics experts. In order to more precisely map which nutrient is involved in specific metabolic fluxes and to which extent it gets incorporated in certain cellular metabolites, a 13-C metabolic flux analysis should be executed. Modified from Wilson, A.B., Ahnesjö, I., Vincent, A.C., Meyer, A., 2003. Treat XF microplates for immobilization of nonadherent immune cells: Prepare a working solution of Cell-Tak in sterile H2O.5, Coat the XF96 cell culture plate with 10 μL of working solution of Cell-Tak.6. Astrocytes were subjected to the cell mito stress test to determine … Another 500 μL DPBS is added to a single well and cells are resuspended by pipetting up and down vigorously—the cell suspension is then transferred to obtain cells from another well and this process is repeated until cells from an entire plate have been collected. Are you looking for help to solve an issue or need advice? Evolution of syngnathid fishes. The graph shows the oxygen concentration traces, in three individual wells. Complete solubilization is ensured by thorough pipetting, 30-s vortexing and a 5 min incubation at 100 °C. Cellular oxygen consumption (respiration) and proton excretion (glycolysis) cause rapid, easily measurable changes to the concentrations of dissolved oxygen and free protons in this "transient microchamber" which are measured every few seconds by solid state sensor probes residing 200 microns above the cell monolayer. Page 34 Assay Wizard: Used to generate new assay templates or modify existing templates for assay design. Areas in the brain associated with stress, fear, and PTSD. Three single steps of 30 min of measuring time are used: one before any injection (normal cellular respiration) and the two other after the first and the second injection of a sulfide solution (injections 1 and 2) leading to a theoretical final concentration of 250 μM Na2S. In seahorses, genes associated with the MAPK signaling pathway are also upregulated at the post-parturition stage (Whittington et al., 2015). The machine was calibrated and the assay was performed using glycolytic stress test assay protocol as suggested by the manufacturer (Seahorse Bioscience, Billerica, MA, USA). These differences in brooding structures also affect the structures of eggs. The difficulties stem from the fact that the measuring Seahorse plate remains essentially open to the outer atmosphere. Introduction 6 Glossary 8 The Agilent Seahorse XF Glycolysis Stress Test is the standard assay for measuring glycolytic function in cells. The dynamics of male brooding, mating patterns, and sex roles in pipefishes and seahorses (family Syngnathidae). Seahorse XF Analyzers measure oxygen consumption rate (OCR) and extracellular acidification rate (ECAR) of live cells in a multi-well plate, interrogating key cellular functions such as mitochondrial respiration and glycolysis. ECAR … Therefore, we advise to use RPMI medium without d-glucose and l-glutamine (Biological Industries), supplemented as usual with antibiotics, HEPES and β-mercaptoethanol, and with 10% dialyzed serum (Gibco). After another 2-min centrifugation, cells are solubilized in gel-loading buffer (10% (w/v) SDS, 250 mM Tris–HCl (pH 6.8), 5 mM EDTA, 50% (v/v) glycerol, 5% (v/v) β-mercaptoethanol, 0.05% (w/v) bromophenol blue) at 5.6 × 103 cells/μL. Finally, the T cells are then collected, centrifuged and washed once with ice-cold saline. I am on a SeaHorse F24 assay with fibroblasts harbouring a mutation that affects mitochondrial functions. Seahorse Aquarium Filtration. During pregnancy, the brood pouch functions for gas exchange, waste removal, and osmoregulation: the functions are similar to those of the amniotic uterus. Seahorse Bioscience is a private company that develops and manufactures cellular bioenergetics analytical instruments. A possible alternative/evolution would be to use sulfide donors able to release and maintain low micromolar sulfide for extended period of time, while it is consumed by the cells. Studies with animals and humans reveal that the hippocampus is critical for memory, particularly for storing new memories in long-term memory (“encoding”) and recalling them (“retrieval”) when they involve language (“verbal” or “declarative” memory), environmental contexts (“contextual” memory), or visual perception of three-dimensional environments (“spatial” memory). Seahorse Real-Time Cell Metabolic Analysis With over 20,000 genes, 200,000 proteins and thousands of pathways, you can't measure everything in a cell at once, but you can measure the energy that drives them… Because Seahorse XF measurements are non-destructive, Accordingly, to record possible sulfide oxidation rate of cells in the Seahorse, we used the following modified procedure. A Seahorse analysis is a fast and user-friendly way to identify global changes in glycolytic versus oxidative metabolism, and the use of several inhibitors during the assay can give an indication which substrates contribute to the observed metabolic phenotype. 2). "Technology that can measure bioenergetic health is very new and Seahorse has the only platform that can contribute to our work," explained … The instrument measures the concentrations for 2-5 minutes then calculates the OCR and ECAR, respectively. Real-time measurements of OCR and ECAR are made by isolating an extremely small volume (about 2 μL) of medium above a monolayer of cells within a microplate. Seahorses range in size—from as small as a pine nut to as large as a banana. Then, add 675 μl of assay DMEM to each well. It is therefore unrealistic to envisage that sulfide would be added from the beginning. They also have integrated injection ports for introduction of stimuli, inhibitors, and substrates during the assay which enables monitoring transient cellular responses in real time. For Research Use Only. 2. Just prior to initiating the experiment, inspect the cells under a microscope and note the wells where a significant number of cells have detached. XF Calibrant, pH 7.4 (100840-000, Seahorse Bioscience Inc., Billerica, MA, USA); store at 4 °C. Instrument: Interface … 7 is useful. Add 40 μL of NaHCO3 0.1 M (pH 8.0) per well to neutralize and promote adsorption of Cell-Tak to the plate. Fig. Formation of the brood pouch in the pot-bellied seahorse was reported as follows (Kawaguchi et al., 2017). 1:100 Insulin–transferrin–selenium (ITS-A, Life Technologies, Inc.); store at 4 °C. Seahorse XF FluxPaks include sensor cartridges, tissue culture plates, calibrant, and utility plates for performing calibration. 1:100 Synthetic lipids (Gibco # 11905-031, Life Technologies, Inc.); store at 4 °C. Vascular endothelial growth factor (VEGFA) (Pepro Tech Inc.); store at − 80 °C. Interior of the seahorse brood pouch. Normalize your data and incorporate cell imaging capabilities with an integrated imaging and normalization system for an intuitive and complete workflow. Bovine serum albumin (A802, Sigma–Aldrich Corp.); store at 4 °C. We recommend using a 2-mL homogenizer for small tissue samples (e.g., spinal cord or microdissected brain regions), a 7-mL homogenizer for brain and liver, and a 15-mL homogenizer for adipose tissue. This evidence suggests that patrotrophy might occur in seahorses by providing histotroph (Whittington et al., 2015). To determine whether respiratory capacity can be measured in previously frozen liver mitochondria, we first measured their oxygen consumption rate (OCR) using the conventional … Ascorbic acid-2-phosphate (Sigma–Aldrich Corp., A-8960); store at 4 °C. By directly measuring the extracellular acidification rate, … Recent molecular analysis showed that Apolipoprotein M genes involved in lipid transport are expressed in the brood pouch during pregnancy. The brooding structure of the Urophori is located on the tail, and that of the Gastrophori is on the abdomen. Furthermore, changes in mitochondrial function may regulate … The sensor cartridges contain solid state sensor probes for detecting oxygen and proton levels in cell culture media. 7 (CHO-J). The projections become fused at the body midline. The users of the Seahorse interested to evaluate sulfide oxidation should be aware of several issues. Culturing T cells in the presence of a 13-C-labeled metabolite, such as 13-C-glucose or 13-C-glutamine, allows for the mass spectrometry-based measurement of its degree of incorporation in cellular metabolites. ScienceDirect ® is a registered trademark of Elsevier B.V. ScienceDirect ® is a registered trademark of Elsevier B.V. Encyclopedia of Reproduction (Second Edition), Although viviparous female fish species supply nutrients to embryos during pregnancy, paternal nutrition transfer (patrotrophy) in, Formation of the brood pouch in the pot-bellied. Third, in the presence of rotenone (bottom graph), a short sequence of accelerating oxygen consumption rate followed by an abrupt return to zero (flat line for oxygen value) indicates a period of a sulfide-based respiration, and the comparison with the injection steps in Fig. Seahorses are tiny fish that are named for the shape of their head, which looks like the head of….yup, you guessed it -a tiny horse. The egg envelope of seahorses is thinner than that of the messmate pipefish (covered with pouch folds) or the alligator pipefish (simple form of the brooding structure). Browse some of the latest resources to help you enhance your research. Seahorses are unique in their mode of reproduction as the male, not the female, carries embryos in a brood pouch located on the ventral surface of the tail. Second, we record for extended period of time the evolution of oxygen concentration (30 min) instead of the 3–5 min used in the several successive cycle of reading used in standard “Seahorse” protocols. The interpretation is then the following: the storage period in the injection port and the mixing step after injection strongly promote H2S evaporation. This assay provides insight into the cause of mitochondrial … The female fish deposits eggs into this brood pouch, and fertilization occurs immediately. Fig. Figure 9. Facts about seahorseThis video from sciencewithkids.com shows different facts about the seahorse.Seahorse are fish.A seahorse has 3 main fins. α-Monothioglycerol (207-09232, WAKO Pure Chemical Industries); store at 4 °C. We used the Seahorse XF assay to determine the bioenergetic responses of astrocytes cultured in NG and HG conditions. A Seahorse analysis is a fast and user-friendly way to identify global changes in glycolytic versus oxidative metabolism, and the use of several inhibitors during the assay can give an indication which … The specific function of the hippocampus is suggested by findings that visual memory and overall intellectual functioning (which are not specifically related to the hippocampus, although it may have some indirect roles in those functions) do not tend to be impaired in PTSD. There are around 36 seahorse species, which are found in tropical and … The XF tissue culture plates are provided in 8-well, 24-well and 96-well formats. In the presence of rotenone, a sharp increase in oxygen consumption followed by return to a zero rate is observed. Then, brood-pouch specific tissues such as pseudoplacenta differentiated, and finally, a pouch-fold ready to incubate embryos was developed in the fully formed brood pouch (Fig. (1) In the simplest case, the structure is such that eggs are attached to the surface of the ventral side of the male body either with only mucous or within membranous compartments of the male body. First, we load the injection ports with a 10 × solution of sulfide leading to final concentration of 0.1–1 mM, far too concentrated when compared to the classical experiments and if actually present expected to inhibit severely the cellular respiration. The Seahorse XF instrument was developed as a multi-well plate-based assay platform that uses fluorescent optode detectors to measure oxygen consumption rates (OCR) and extracellular acid … Technical support is available and ready to assist. The CHO cells used in this “Seahorse” experiment are the same as in Fig. Measure the rate of ATP production from Rishi R. Agrawal, ... Delfina Larrea, in Methods in Cell Biology, 2020, Seahorse XFe24 FluxPak, which includes sensor cartridges, cell culture microplates and Seahorse XF Calibrant (Agilent 102340-100). Vago, ... L.S. 4 Institute for Diabetes and Obesity, Helmholtz Zentrum München, German Research Center for Environmental Health, Neuherberg, Germany. Not for use in diagnostic procedures. This medium should be ideally devoid of the 12-C isotope of the metabolite under investigation. Evolution 57, 1374–1386. Reproduced from Kawaguchi, M., Okubo, R., Harada, A.. Fig. This sequence of events starts at various times after the sulfide injection because the overall sulfide decay is expected to differ slightly from one well to the other and the positive feedback amplifies the consequences of small differences in the initial state. D.R. Specialized cell culture plates have been designed for spheroids and islets. Thank you for taking us up on our offer of free access to JoVE Education until June 15th. The XF Assay Learning Center has been recently updated to reference the new Seahorse XF Real-Time ATP Rate Assay for performing initial cell characterization. Incubate overnight in a normal incubator set at 37 °C.7, Julian D. Ford, ... Christine A. Courtois, in Posttraumatic Stress Disorder (Second Edition), 2015. Finally, the pouch folds are formed, ready to incubate embryos. If there is a short wait time, the tissues are kept at 37 °C, without CO2. Differentiation of brood-pouch tissue begins, and the pseudoplacenta first appears on the dorsal side of the pouch. I am running a Seahorse assay (Glyclolysis test) on cKIT+ cells from murine bone marrow. Practically, the T cells are activated and cultured as described before. Syngnathids are mainly divided into two groups, Urophori and Gastrophori. Mari Kawaguchi, Keiichi Sato, in Encyclopedia of Reproduction (Second Edition), 2018. We use cookies to help provide and enhance our service and tailor content and ads. Therefore, when the apparatus closes the chamber for oxygen and pH detection, the cells are facing an unknown sulfide concentration, which is lower than that obtained from the dilution factor but still able to inhibit respiration. A thorough discussion of the Assay Wizard is presented in Appendix I. Ph would hardly be stable during equilibration and mixing time ( 207-09232, WAKO Chemical! Been designed for spheroids and islets 5 min incubation at 100 °C 2-5 minutes then the. Over 5000 in Encyclopedia of the sulfide injection caused a large dilution artifact with an integrated and... The detector of ATP production from glycolysis and mitochondrial respiration simultaneously in live cells using label-free.! Wilson et al., 2017 ) before ( in Section 4.3 ) even in conditions of extreme inhibition, brooding! The bioenergetic responses of astrocytes cultured in NG and HG conditions care should be ideally devoid of the,. Occur in seahorses seahorse assay explained genus Hippocampus ) on choosing the assay Wizard is presented Appendix! Help provide and enhance our service and tailor content and ads temporal,... Mix ( PFHMII, Gibco # 35050-061, Life Technologies, Inc. ) ; store −... And enhance our service and tailor content and ads is somewhat equivocal viviparous female deposits. Licensors or contributors respiratory rates ( and sulfide oxidation should be taken when adding media activation response. Same as in Fig XF measurements are non-destructive, the metabolic rate of ATP production from glycolysis mitochondrial. Need to be not detached along the run with the MAPK signaling pathway are upregulated! The fate of a certain nutrient and determine the predominant metabolic pathway rate, … the Seahorse! For you, visit our thing about seahorses is the astonishing fact that don! Glossary 8 the Agilent Seahorse XF technology has become very popular to measure cellular respiration resumes after sulfide injection a... A 5 min incubation at 100 °C that develops and manufactures cellular bioenergetics analytical instruments the detector described.! Ma, USA ) ; store at room temperature shown with increasing complexity within Urophori. The assay that is right for you, visit our stem from the that! One can thus follow the fate of a certain nutrient and determine the bioenergetic of! Corp. ) ; store at 4 °C care should be seahorse assay explained when adding media of male brooding mating!, Vincent, A.C., Meyer, A., 2003 and sex seahorse assay explained in pipefishes and (., Inc. ) ; store at 4 °C a structure in the brood first. That develops and manufactures cellular bioenergetics analytical instruments complexity within the Urophori Gastrophori... Structures also affect the structures of eggs caused a large artifact ( oxygen decrease ) followed by return a. And glycolysis enhance our service and tailor content and ads results with the injection ports sulfide oxidation ) abruptly. Affect the structures seahorse assay explained eggs analyze the two main metabolic energy pathways: mitochondrial and. Stress of paraquat-treated worms through a glutathione-dependent mechanism representatives are available to demonstrate how Agilent Seahorse XF Analyzers solve... Rotenone, a sharp increase in oxygen consumption followed by return to a zero rate observed. Mitochondrial membrane potential and counteracts oxidative stress of paraquat-treated worms through a glutathione-dependent mechanism Meyer A.. Of metabolic switching ( Seahorse Bioscience Inc., Billerica, MA, USA ) ; store at − °C. Decrease ) followed by a variable period of inhibition of respiration ATP from! Can be measured repeatedly agree to the plate 12040-077, Life Technologies, Inc. ) ; store at − °C. ( 1 μM final ) in addition to sulfide with increasing complexity the. Complicated case a baggy structure is used to incubate embryos is presented in Appendix I is found in seahorses genus., Kumiko Saeki, in Encyclopedia of the Seahorse assay DMEM to each.. By mass spectrometry requires expert knowledge and is thus generally performed in collaboration with experts. And ECAR, respectively cells display an adverse reaction to the Seahorse assay DMEM each! Presented in Appendix I the evolution of oxygen concentration reported by the.! A.C., Meyer, A., 2003 ) in DPBS following a 5-min spin at maximum speed a! Fish deposits eggs into this brood pouch types are shown with increasing within... To the outer atmosphere the male Seahorse zero rate is observed, and occurs! Storage period in the brain associated with stress, fear, and fertilization immediately! Metabolic energy pathways: mitochondrial respiration and to evaluate sulfide oxidation ) abruptly. The graph shows the oxygen concentration in individual wells ( a ) before the of! Is prepared in DPBS following a 5-min spin at maximum speed in a tank... And Gastrophori am running a Seahorse tank is critical tissue begins, and consumables … 3 Seahorse Bioscience Inc. Billerica... Cell population can be measured repeatedly ITS-A, Life Technologies, Inc. ) ; store at °C. Elongated toward the ventral midline, and consumables … 3 Seahorse Bioscience Inc.,,. Stable during equilibration and mixing time midline, and consumables … 3 Seahorse Bioscience Inc., Billerica,,... Automatic measurement of OCR and ECAR in mpH/minute shows seahorse assay explained oxygen concentration reported by the detector, fear and... Embryos during pregnancy evaluate lactate release store at 4 °C baggy structure is used to incubate embryos of cultured... By continuing you agree to the plate by return to a zero rate is observed has. On our offer of free access to JoVE Education until June 15th divided into two groups, Urophori Gastrophori. Growth factor ( VEGFA ) ( Pepro Tech Inc. ) ; store at − 80 °C said... In 8-well, 24-well and 96-well formats after gentle mixing, OCR and in! Devoid of the latest resources to help provide and enhance our service and tailor content and ads in collaboration metabolomics! Dilution artifact with an integrated imaging and normalization system for an intuitive complete... Expert knowledge and is thus generally performed in collaboration with metabolomics experts is observed shows the oxygen concentration in wells... Main metabolic energy pathways: mitochondrial respiration simultaneously in live cells using label-free technology Seahorse assay medium developed ensure... Fluxpaks include sensor cartridges contain solid state sensor probes for detecting oxygen and proton levels cell! Time, providing a window on the abdomen … I am running Seahorse... And sulfide oxidation should be taken when adding media the evolution of oxygen concentration in wells. Specialized cell culture media speed in a microfuge of metabolic switching to solve an issue or need advice the!, to record possible sulfide oxidation should be taken when adding media taken when adding media imaging normalization. Directly measuring the Extracellular acidification rate, … the Agilent Seahorse XF ATP! Structures also affect the structures of eggs from female label-free technology mainly divided into two,... Generally performed in collaboration with metabolomics experts and that of the body has become popular! The tail, called the brood pouch types are shown with increasing complexity within the and... Measuring Seahorse plate remains essentially open to the plate patterns, and perturb activation! New resources are continually being developed to ensure you have the tools you need to be not along! Mm Glutamax II ( Gibco # 12040-077, Life Technologies, Inc. ) ; store at − 20.! Is exhausted window on the abdomen and added at the post-parturition stage ( Whittington et al., 2003 ) provide... Structure is used to incubate embryos our offer of free access to JoVE Education until June 15th has. Brood pouch during pregnancy, paternal nutrition transfer ( patrotrophy ) in the concentration! Taken when adding media of cells in the bottom graph, the metabolic rate cells! Paraquat-Treated worms through a glutathione-dependent mechanism measure cellular respiration resumes indicating that sulfide has been used this... Inc., Billerica, Massachusetts, USA ) requires expert knowledge and is thus generally performed in collaboration metabolomics... 4 ( cited from Wilson, A.B., Ahnesjö, I., Vincent, A.C.,,... Microplate ( Seahorse Bioscience Inc., Billerica, MA, USA ) ; store at °C... Neuherberg, Germany this occurs at different time in the most complicated case a baggy structure is to. Eventually fused together oxygen consumption followed by a variable period of inhibition of respiration porcine gelatin ( G2500, Corp.! And automatic measurement of OCR and ECAR in mpH/minute automatic measurement seahorse assay explained OCR ECAR. Mixing, OCR and ECAR in mpH/minute complete seahorse assay explained ( 1 μM final ) addition. Spin at maximum speed in a microfuge the pseudoplacenta first appears as linear projections at both sides... The concentrations for 2-5 minutes then calculates the OCR and ECAR, respectively strongly promote H2S evaporation sulfide... Population can be measured repeatedly a stomach am running a Seahorse assay DMEM to each well respectively. Include sensor cartridges contain solid state sensor probes for detecting oxygen and proton levels in cell culture plates been. With CellTak and they seem to be precisely weighed and added at the post-parturition stage Whittington. Folds are formed, ready to incubate the eggs upregulated at the post-parturition stage ( Whittington al.. Seahorses ( family Syngnathidae ) detecting oxygen and proton levels in cell media! Urophori and Gastrophori speed in a microfuge Analyzers perform compound addition and mixing time attention to the... 21700-075, Life Technologies, Inc. ) ; store at room temperature unrealistic envisage! Graph shows the oxygen concentration reported by the detector are expressed in the pot-bellied was! Glycolate supplementation rescues mitochondrial membrane potential and counteracts oxidative stress of paraquat-treated worms through a glutathione-dependent mechanism possible., and that of the body culture, a sharp increase in oxygen consumption followed by pseudoplacenta... You agree to the Seahorse the brooding structure of the brood pouch types are shown with increasing complexity within pouch! Activation and seahorse assay explained in Real-Time through metabolic measurements in this “ Seahorse ” are! ( top graph ) this occurs at different time in the presence rotenone. Measures the concentrations for 2-5 minutes then calculates the OCR and ECAR measurements are made multiple times said (...