Seahorse XF96 Cell Mito Stress Test protocol to measure mitochondrial respiration • Highlights the critical steps to be considered during the experiment • Lists the limitations and problems with the Seahorse XF96 Cell Mito Stress Test Summary Mitochondria play pivotal roles in cellular energy metabolism. 1. AD-MSCs and BM-MSCs displayed higher mitochondrial transfer than DP-MSCs and WJ-MSCs. 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. Oxygen consumption rate (OCR) was assessed by the use of a Seahorse Extracellular Flux (XFe24) Analyser with the XF Cell Mito Stress Test Kit (Seahorse Bioscience Inc. North Billerica, MA) based on a previous study (Yang et al., 2017). mitochondria in the XF24 Analyzer George Rogers, PhD, Seahorse Bioscience Anne Murphy, PhD, University of California, San Diego Alvaro Elorza, PhD, Andres Bello University Martin Brand, PhD, The Buck Institute for Age Research Technical Brief Prior to Day of Assay Day of Assay Prepare MAS & substrates (succinate, malate) Isolate and seed mitochondria in MAS + substrate Prepare stock compounds . The Seahorse XF Cell Mito Stress Test is the gold standard assay and is widely used for measuring mitochondrial function in cells. Recent studies have demonstrated mesenchymal stem cells (MSCs) as effective mitochondrial donors with therapeutic success in multiple experimental models of human disease. MAS-3: KCl based MAS: Seahorse Bioscience has used this buffer with success for mitochondria purified from mouse liver and rat liver. Mitochondrial respiration results from the transfer of electrons between complexes I, II, III, and IV, with complex IV reducing oxygen to water; thus, oxygen consumption integrates electron transport activity from complexes I/II through IV. Cellular energy metabolism research is highly topical in all fields of mammalian cell biology. Mitochondrial respiration in Caco-2 and HepG2 cells was characterized as an indicator of cellular metabolism and fitness in response to the exposure to CER by extracellular flux analysis using Agilent Seahorse XF24 Analyzer (Agilent Seahorse Bioscience, Santa Clara, CA, USA). Total intracellular adenosine triphosphate (ATP) is mainly generated by glycolysis and mitochondrial oxidative phosphorylation. These publications offer a direct comparison to Clark-type electrodes in models normally measured by this method. Squirting the mitochondria through narrow pipette tip or pipetting with force may rupture the mitochondria. Coupling Assay Delfina Larrea, in Methods in Cell Biology, 2020 3.1 Materials • After the completion of the run, monitor the pH changes by selecting pH on Y2-axis in the overview mode. Notably, recent publications highlight the application of the Seahorse XF24 to the study of isolated mitochondria in a more high-throughput fashion [8, 9]. Little, Kovalenko et al. Use of intact cells can provide information in the context of ATP demand as. In mammalian cells, glycolysis and oxidative phosphorylation (OXPHOS) pathways provide the majority of cellular ATP. and respiratory capacity in a single experiment to assess mitochondrial dysfunction. Importantly, data derived from the Seahorse XF24 instrument compare favorably to those acquired using the Clark-type electrode . this buffer tends to be more protective of isolated mitochondria. The chamber is then unsealed (allowing. Add the following solutions (these are standard concentrations and may be slightly different for your cell line of interest): 10 mM Glucose (1 mL of a 1M stock into 100 mL) 2 mM Glutamine (1 mL of a 200 mM stock) 1 mM Pyruvate (100 uL of a 1M stock) Seahorse Bioscience is a private company that develops and manufactures cellular bioenergetics analytical instruments. MSCs obtained from different tissue sources such as bone marrow (BM), adipose (AD), dental pulp (DP), and Wharton's jelly (WJ) are routinely used in clinical trials with no known study of their mitochondrial donor capacity. While OXPHOS consumes O. For adherent cells I would use Seahorse, for mitochondrial experiments either Seahorse or oxygen electrode (eg Oroboros). In contrast, the Seahorse analyzer assay with its sensor cartridge system enables measurement of mitochondrial respiration and glycolysis in real time and in a non-invasive manner that does not require any dyes or labels. Respirometry assay using conventional Seahorse protocol Mitochondria and homogenates were loaded into Seahorse XF96 microplate in 20 μl of MAS containing substrates. 2. During Seahorse mitostress assays, we have great variance between different cell lines regarding their oxygen consumption. Overnight incubation of the cartridges in the calibration buffer at 37 °C in a non-CO 2 incubator is highly recommended. Seahorse 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. Oxygen consumption rate (OCR) is measured before and after the addition of inhibitors to derive several parameters of mitochondrial respiration. Initially, baseline cellular OCR. Counterintuitively, DP-MSCs and WJ-MSCs were more effective in suppressing mtROS levels in stressed recipient cells . Mitochondria (5 µg/well mouse liver mitochondria or 2-3 μg rat heart mitochondria work well in the Seahorse XFe/XF24, respectively for succinate) begin in a coupled state with substrate present, in this case, succinate (10 mM), and rotenone (2 µm) (State 2). The Agilent Seahorse XF ATP Real-Time rate assay measures and quantifies the rate of ATP production from glycolytic and mitochondrial system simultaneously using label-free technology in live cells. The Seahorse system, for instance, utilises solid-state fluorescence-based sensors to measure extracellular O 2 levels within a sealed chamber for 2−6 min. It helps to find out about . Company President and CEO, Jay Teich founded the company in 2001 along with Andy Neilson and Jim Orrell. Place 100mL in non-CO2, humidified, 37C incubator for ~30 minutes to warm up. Reference couple the metabolic flux assay from the Seahorse bioanalyzer with high-content fluorescence imaging to study the coordinated biology of mitochondrial bioenergetics and . Cells were plates at 4 × 10 4 . . Seahorse XFe24 experiment. From: Placenta, 2019 Download as PDF About this page Mitochondria, 3rd Edition Rishi R. Agrawal, . Experiments performed by Seahorse Bioscience have shown that comparable results have been obtained using either MAS with . The plate was then transferred to the Agilent Seahorse XFe/XF96 Analyzer, and the experiment initiated. This buffer is more representative of the physiologic conditions in vivo. Using Seahorse Machine to Measure OCR and ECAR in Cancer Cells A large amount of energy used for nutrient processing and cellular functions is essential for tumorigenesis. Experimentally distinct, but parallel, experimental approaches are used to quantify the potential and flux components of the proton circuit, both with isolated mitochondria and intact cells. Mitochondrial reactive oxygen species (mtROS) levels were analyzed by using MitoSOX red-based staining, and mitochondrial respiration parameters were analyzed by using a Seahorse XF Analyzer. Introduction Agilent Seahorse XF Cell Mito Stress Test Kit User Guide 7 Carbonyl cyanide-4 (trifluoromethoxy) phenylhydrazone (FCCP) is an uncoupling agent that collapses the proton This assay provides insight into the cause of mitochondrial dysfunction and an in-depth understanding of metabolic pathways, signals, and phenotypes. 2, driving the oxygen consumption rate (OCR), both . (A) Parameters of mitochondrial respiration were derived by the sequential addition of pharmacological agents to respiring cells. The Seahorse Assay and Experiment Timeline. Seahorse XF analyzers (marketed by Agilent) are being used to assess mitochondrial function by measuring their oxygen consumption rate (OCR) in response to modulators of key components of the. The Agilent Seahorse Cell Mito Stress Test kit is the widely recognized, well accepted standard assay for assessing mitochondrial function. The mitochondria were viewed briefly under a microscope at 20x to ensure consistent adherence to the well. Multiple parameters are obtained in this one assay including, basal respiration, ATP-linked respiration, maximal and reserve capacities, and non-mitochondrial respiration. The fluorescence of 1 × 10 4 cells for each experiment was quantified with a flow cytometer (CytoFLEX S, Beckman Coulter . (1) The pmf is expressed in mV. This proton circuit is central to mitochondrial bioenergetics. Seahorse Bioscience is headquartered in North Billerica, Massachusetts, with its . The Seahorse assay. 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