Oligomycin seahorse. Seahorse uses a series of injections to add the modulators of respiration to assess the mitochondrial function (Fig. In the measurement of OCR, oligomycin, FCCP, and rotenone/antimycin A are sequentially utilized. Aliquots in the -20 in Seahorse Reagents box. The following table summarizes the recommended starting concentrations for the compounds in the Seahorse XF Cell Mito Stress Test and the key parameters derived from the resulting OCR measurements. The injection of oligomycin, which, by inhibiting ATP synthase, could rapidly hyperpolarize the mitochondrial membrane, thereby preventing protons passing through the complexes. As it is mentioned above, a dose response on seahorse like FCCP titration would be good to try. This instruction sheet describes a basic procedure for optimizing FCCP concentration using the Agilent Seahorse XF Cell Mito Stress Test with XF Pro analyzer. Any Seahorse experts out there? Difficulty with Oligomycin Hi All, I'm currently working on learning the Agilent Seahorse Mitochondrial stress assay, and facing one major difficulty -- I can't get my Oxygen Consumption Rate (OCR) to plateau upon injection of Oligomycin. The XF Cell Mito Stress Test is run with five different FCCP concentrations to determine the optimal concentration for use in XF assays, as indicated in Figure 1A. We would like to show you a description here but the site won’t allow us. In a typical Seahorse experiment, the first compound to be added is Oligomycin, which inhibits ATP synthase (complex V) by blocking the proton translocation in the FO part. Next oligomycin, an inhibitor of complex V, is added, and the resulting OCR is used to derive ATP-linked respiration (by subtracting the oligomycin rate from baseline cellular OCR) and proton leak Oligomycin A or the complex of ABC? Looking to buy some oligo for my seahorse assay from a third party vendor to save $, just wondering if it is oligmycin A I want or the complex. Oligomycin inhibits ATP synthase (complex V), and is injected first in the assay following basal measurements. A lack of response to Oligomycin, an inhibitor of ATP synthase (Complex V), is a common issue observed in the Seahorse XF Cell Mito Stress Test. In the model, the Oligomycin should flatline OCR. For use with Seahorse XF HS Mini and XFp analyzers. This technical support center provides troubleshooting guides and frequently asked questions (FAQs) to help researchers, scientists, and drug development professionals interpret Seahorse XF assay data with an abnormal response to Oligomycin A. Sep 2, 2025 · Seahorse - Mitochondrial Stress Test Materials Seahorse XF Media XF Cartridge Cells in XF24 plate, either grown in the plate or seeded at a previously established density where the OCR is 40-500 mL/min. Oligomycin, an inhibitor of ATP synthase, allows the measurement of ATP-linked respiration. Injection stock solutions (1 mM Oligomycin, 1 mM FCCP, 1 mM Rotenone and 1 mM Antimycin A) made up in DMSO at 1000X. Oligomycin works by binding to ATP synthase and blocking the ion channels. This document provides a detailed protocol for performing a Seahorse XF Cell Mito Stress Test on adherent cells, with a specific focus on the role and application of Oligomycin A. . 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. It impacts or decreases electron flow through the ETC, resulting a reduction in mitochondrial respiration or OCR. Hydrate a cartridge by placing 1 mL/well of XF Calibration Media into each well of a XF24 cartridge. Check if the machine is free. The optimization step involves drug optimization (eg, oligomycin, FCCP) and optimization of sample quantity (eg, cell seeding number, quantity of mitochondria). 1). Targets of the drugs utilized in the Seahorse assay. Seahorse XFp Real-Time ATP Rate Assay Kit. Each pouch contains one (1) each of oligomycin and rotenone/antimycin A. This is characterized by the failure of the Oxygen Consumption Rate (OCR) to decrease after the injection of oligomycin. The Seahorse XF Glycolysis Stress Test Kit includes: Six foil pouches each containing oligomycin Six vials containing glucose Six vials containing 2-DG The kit reagents are sufficient for six complete XF Glycolysis Stress Test assays in a 96 or 24-well Seahorse XF Cell Culture Microplate. Another thing you can Sep 2, 2025 · Injection stock solutions (1 mM Oligomycin, 1 mM FCCP, 1 mM Rotenone and 1 mM Antimycin A) made up in DMSO at 1000X. Mar 19, 2021 · Materials and equipment Note: The Cell Mito Stress Test Kit contains oligomycin, carbonyl cyanide-4- (trifluoromethoxy)phenylhydrazone (FCCP) and rotenone/antimycin A. Aug 1, 2024 · The Seahorse assay process includes steps from optimization of conditions to data interpretation. Below are potential causes and solutions to troubleshoot this problem. Each kit containing six (6) single-use pouches. Nov 28, 2022 · Oligomycin inhibits ATP synthase (complex V), and is injected first in the assay following basal measurements. nmw kge efr qxe zzz eaa kwk isj pfp iwj gex ptk sgl upu pac