). Isolated Mitochondria Assay Flow Chart . 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. (a) The two graphs show time-dependent changes in OCR and ECAR measured sequentially injecting oligomycin, FCCP, and rotenone plus antimycin A on primary cortical neurons of wt and α-syn null mice in basal conditions or after rotenone exposure. I always plate each well with same cell number. Mitochondrial respiratory profiles from Seahorse assays on isolated brain mitochondria performed in our laboratory. ab109903. Although cells can be offered various substrates in the assay medium, many cell types can oxidize stored pools of energy substrates. 103344-100 and Cat No. MitoProbe DiOC 2 (3) Assay Kit; Readout: Active mitochondria exhibit brighter red fluorescence signal compared to mitochondria with lower membrane potential which fluoresce green. Mitochondrial Architecture. Notes. Agilent Seahorse XF Mito Fuel Flex Test Kit (Cat No. 102504-100) Agilent Seahorse XF Glycolytic Rate Assay Kit (Cat No. Respirometry assay using conventional Seahorse protocol. We adapted primary cultures of renal proximal tubular cells (RPTCs) that exhibit in vivo levels of aerobic metabolism, are not glycolytic, and retain higher levels of differentiated functions and used the Seahorse Bioscience analyzer to measure mitochondrial function in real time in … 103346-100) XF Fatty Acid Oxidation Profile Working with isolated mitochondria is the gold standard approach to investigate the function of the electron transport chain in tissues, free from the influence of other cellular factors. Complex immunocapture. Thus, to directly investigate the reduction of mitochondrial proton leak in old cardiomyocytes by SS-31, we expressed the protein mt-cpYFP, a mitochondrial matrix-targeted pH indicator (Demaurex and Schwarzländer, 2016; Schwarzländer et al., 2012; Wang … Enable user to develop own assays based on captured complex. Mitochondria and homogenates were loaded into Seahorse XF96 microplate in 20 μl of MAS containing substrates. The experiment was performed as explained in Section 5. Two milligrams of mitochondria per assay were used, and 40 μM pulses of CaCl 2 were continually given until mitochondrial Ca 2+ uptake no longer occurred or the mitochondria underwent MPT as evidenced by irreversible increase in Calcium Green-5N fluorescence, which was saturated in the presence of 40 μM alamethicin (arrowhead). As such, assays that collect direct measurements of drug-related effects on mitochondrial respiration have gained interest from industry due to their increased sensitivity. Capture complex and assay activity. CONTRACT RESEARCH SERVICES. drug candidate, etc. Mitochondria are diluted into 1X MAS containing the substrate of choice. Toxicity assay. ... Key features: Compatibility with both adherent and suspension cells as well as isolated mitochondria. The Seahorse assays were performed in Seahorse XF DMEM supplemented with glucose to a final concentration of 10 mmol/L, sodium pyruvate to 1 mmol/L, and l ‐glutamine to 2 mmol/L. Significantly, we show that mitochondria supply ATP to the ER and a SERCA-dependent Ca 2+ gradient across the ER membrane is necessary for ATP transport into the ER, ... XF24’s standard operating procedures as per manufacturer’s manual, facilitated by a XF24 extracellular flux assay kit (Seahorse BioSciences, Part # 100850–001). Analyzing whole islets has the advantage of maintaining the cellular architecture. A general bioenergetic profile can therefore be obtained using intact cells, but the inability to control substrate provision to the mitochondria can restrict an in‐depth, mechanistic understanding. ab109904. Bioenergetics measurements were performed, after sequential treatment with Oligomycin, FCCP, and R+A (Agilent Technologies), at 2 , 7 , 14 , and 28 days post‐transplantation and compared … The results were analyzed using the Seahorse software, and the change in OCR and ECAR before and after Aa injection was calculated to assess the cells’ metabolic potential. Immunocapture activity assay. pone-0085436-g001: The Seahorse assay.Oxygen consumption rate (OCR) is measured before and after the addition of inhibitors to derive several parameters of mitochondrial respiration. ab109711. In this chapter, we outline a detailed protocol to measure the rate of oxygen consumption (OCR) with the high-throughput analyzer Seahorse XF96. However, as described here for cellular assays, robust normalization procedures require careful analysis. Intact Cell. The Agilent Seahorse analyzers utilize patented transient micro-chambers and embedded fluorophores to make rapid, sensitive, and precise metabolic measurements in real time. Complex II. We will outline the thresholding and other qualification measures we employ to the MitoXpress Xtra Oxygen Consumption assay and to the Agilent Seahorse XFe96 analyzer with the Cell Mito Stress Test. This oxygen-sensitive phosphorescent probe can be used to assess O 2 consumption by cultured cells, isolated mitochondria, microorganisms, tissues, and enzymes.. Phosphorescent probe increases in signal intensity with O 2 consumption ( O 2 levels); Amenable to 96- and 384-well microplates for high-throughput analysis on … Here, we describe two high‐throughput assays, one for assessing fatty acid oxidation in cells and the other for assessing fatty acid oxidation in isolated rat liver mitochondria. 103260-100 and Cat No. Even if you are an experienced user of isolated mitochondria, Seahorse In addition, there are no high-throughput real-time assays that assess mitochondrial function. MITO-ID ® Extracellular O 2 Sensor Kits . The BCA assay was performed according to the manufacturer’s instructions, and the protein levels were used to normalize the Seahorse data for each individual well. Mitochondria can be interrogated and characterized in novel ways, looking at rates of substrate metabolism, sensitivity to drugs and other chemicals, and effects of mutations in mitochondria-related genes. But they could still be different after 2-3 days of culturing. 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. An adaptation of the Seahorse XF plate assay has been the development of microplates that are capable of analyzing pancreatic islets for the study of insulin secretion . Mitochondrial Respirometry . Initial OCR was measured to establish a baseline at the resting state (basal respiration) followed by injection of oligomycin (an inhibitor of ATP synthase) … In addition, the Seahorse XF Analyzer can be used on isolated mitochondria and on isolated enzymes, where a normalization approach is to divide OCR by mitochondrial proteins or enzyme concentration . Lysosomes are pivotal in regulating metabolism, endocytosis and autophagy and are implicated in cancer. 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