fccp titration seahorse

Prior to plating, Seahorse V7 tissue culture plates were coated with 20 μg/ml laminin (Invitrogen mouse laminin, 23017) for 2 h at 37°C. FCCP, carbonyl cyanide 4-(trifluoromethoxy)phenylhydrazone. Spin down mitochondria at 4°C for 10 . II.1. II.1.1. Cell-based respirometers, such as the Seahorse Extracellular Flux Analyzer, are valuable tools to assess the functionality of mitochondria within adherent neurons, as well as other cell types. Retinal photoreceptors are the highest energy-consuming cells in the body, requiring abundant energy substrates. . Download Download PDF. A andB: BeWo cells were incubated at 37°C for 10 min in uptake buffer containing 0.5% ethanol with or without 50 μM FCCP. Peroxisome proliferator activated receptor-alpha (PPARα) is a ubiquitously expressed nuclear receptor. Compatibility . Normalizing to mitochondrial content after running a Seahorse experiment (Fig. By titration experiments, we determined the accuracy to be approximately ±20%-25% (data not shown). Synaptosomes were incubated in the presence of 15 mM glucose + 10 mM pyruvate and the indicated concentra-tions of inhibitors were added to model mitochondrial dysfunction found in Par- II.1.1. The focus of this software release is to provide a simple, straightforward Seahorse data analysis experience through a more intuitive user interface that supports routine functions for all Seahorse XF Analyzer data. Multiple titrations with FCCP are not possible during the experiment without compromising other additions such as oligomycin, Antimycin+Rotenone. 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 Bioscience Asia 199 Guo Shou Jing Road Suite 207 Pudong, Shanghai 201203 CN and its slowdown after FCCP further indicates the dependence of cell on glycolysis and the increase of lactate production. The XFe-24 flux analyzer measures real time kinetic of the media flux in live cellular environment to determine in vitro oxygen consumption rate (OCR), and extracellular acidification rate (ECAR), in order to assess cellular metabolic functions such as oxidative phosphorylation, glycolysis, and fatty acid oxidation. Andrew Knight. Significant positive association were seen FCCP-stimulated flux normalized to cell count (A) and protein concentration (C) and represented as percentage of basal respiration (B). As expected, the concentrations of FCCP (median concentration of 5 μM) required to achieve maximal OCR in T98G cells were lower ( P <0.01) ( S3D File ) than those required . Specific titration steps in the protocol disrupt the physiological control of respiration, which then allows for . FCCP titration in the presence of oligomycin in RAW 264.7 macrophage cells. Seahorse XFp cartridges have only four injection ports and therefore allow only four injections in the well (size similar to the 96-well plate wells) during the experiment. Oxygen consumption was measured using a Seahorse XF96 Flux Analyzer on mitochondria respiring on pyruvate (10 mM) and malate (2 mM) or succinate (10 mM) and rotenone (2 μM). on PCs (Personal Computers - Laptop or Desktop) with a Windows Figure 2 Respirometry measurements of complex I, II, and IV comparing fresh versus frozen tissue proto- cols. The Seahorse Extracellular Flux XF24 analyzer provides a comprehensive assessment of the relative state of glycolytic and aerobic metabolism in live cells making this method instrumental in assessing mitochondrial function. FCCP titration in the presence of oligomycin in RAW 264.7 macrophage cells. Wave Desktop 2.3 is intended for use . Mouse liver mitochondria were isolated and respiration was measured according to Rogers et al. Divide into aliquots and then store at -20 °C. More recently, a gestational high fat diet independent of maternal body composition, has been highlighted as a potential independent regulator of placental mitochondrial metabolism . Multichannel pipettes and tips . RAW 264.7 macrophage cells were seeded into Xr96 microplates (Seahorse Bioscience) at a density of 8.0 x 104 cells/well. Preparation of 5x KHB base from dry reagents . II. concentration titration experiment is recommended for a specific cell type/line. The role of endogenous PPARα in retinal neuronal homeostasis is unknown. The above concentration of oligomycin, FCCP, antimycin, and rotenone apply to all figures in this manuscript. If you truly want to compare max respiration for each different cell line (eg. To optimize analysis of human PBMCs or CD4 + T cells (~700,000 cells per well), we first titrated the ATP synthase inhibitor oligomycin and the mitochondrial uncoupler FCCP from 0-4 μM, and the complex I/III inhibitors rotenone and antimycin A, respectively, from 0-14μM using the Seahorse XF Mito Stress Test program per manufacturer's . Multichannel pipettes and tips . For Seahorse metabolic proﬁling, cells were plated at 5 . FCCP Titration in the Presence of Oligomycin in RAW 264.7 Macrophage Cells RAW 264.7 macrophage cells were seeded into XF e 96 microplates (Seahorse Bioscience) at a density of 8.0×10 4 cells/well. Hepatocellular carcinoma (HCC) is the most prevalent form of liver malignancy and carries poor prognoses due to late presentation of symptoms. (A) The Seahorse traces and associated OCR (oxygen consumption rate) quantiﬁcations of mouse liverhomogenates.hFresh represents liver homogenate which was isolated and tested without being frozen.TMPD is added to a subset of the hFresh samples to uncouple the mitochondria to survey . Optimal concentrations of FCCP were determined by monitoring OCR during FCCP titration (Figure (Figure1F). The Angio BioCore (ABC) is a state-of-the-art facility that has been established to conduct validated and highly reproducible in vitro and in vivo angiogenesis, hematopoietic and multi-parametric flow cytometry assays. Titration of basal, coupled [oligomycin-sensitive] and maximal [FCCP-stimulated] respiration with (a) rotenone and (b) 3-NPA. drial respiration. All the dilutions were freshly prepared. Compared with the values for T98G cells treated with the vehicle DMSO before FCCP titration, SRC was underestimated by 36.7 ± 1.8% in T98G cells treated with oligomycin . III.6 . Prepare 50X or 100X stock of sodium phosphate (monobasic and dibasic) stock . FCCP Sigma C2920 0.4 to 4 µM Stock 10000X (4-40 mM) in DMSO Dilute to 10X in MAS . It is strongly suggested to perform a titration experiment with the mitochondria to determine the optimal amount to use. FCCP titration assay was applied to determine the optimal FCCP dosage by continuously injecting FCCP until the decreased OCR value was reached in mouse neonatal cardiomyocytes (data not shown). Download figure Download PowerPoint Preparation of 5x KHB base from dry reagents . The maximum respiratory rate is caused by injection of uncoupler FCCP, so if OCR is not increased following the FCCP injection, you should perform a careful titration of FCCP to optimized the appropriate concentration. Then, the following final concentrations of the inhibitors were added after a set of titration experiments: 24 µg mL −1 oligomycin, 0.8 × 10 −6 m FCCP, 5 × 10 −6 m rotenone, and 15 × 10 −6 m antimycin A. Compared with the values for T98G cells treated with the vehicle DMSO before FCCP titration, SRC was underestimated by 36.7 ± 1.8% in T98G cells treated with oligomycin . Mitochondrial titration assays. Once you have your cell number optimized, do another titration for FCCP. C: carbonyl cyanide-4- (trifluoromethoxy)phenylhydrazone (FCCP) titration, OCR normalized to last time point before addition of FCCP ( left ), maximum OCR in each well after addition of FCCP ( right ), error bars indicate means ± SE for 5-6 replicates and P values calculated by one-way ANOVA. In brief, basal OCR measurement was followed by measurements taken after sequential addition of oligomycin, FCCP, and rote-none/antimycin A. ATP turnover was defined as (last rate measurement before oligomycin) − (minimum rate measure-ment after oligomycin injection) (27). This duality of MYC underscores the importance of further investigation into its role in SCLC metabolism and could lead to insights into metabolic targeting approaches. Figure 2 Respirometry measurements of complex I, II, and IV comparing fresh versus frozen tissue proto- cols. **Cells that have been shipped overnight on wet ice (not cold packs as the cells can freeze!) concentration titration experiment is recommended for a specific cell type/line. Media was changed to unbuffered DMEM, 20% FBS, 200 mM Glutomax (Thermofisher Scientific Cat# 35050061), 100 mm sodium pyruvate, 25 mm glucose, and pH 7.4. The FCCP concentration required for maximal respiration was established by titration (data not shown) using osteocytes from control mice. Select XF Cell Mito Stress Test on the Templates window and set up the program as follows: b. Using a Seahorse analyzer to measure the oxygen consumption rates . (E-I) Confocal microscopy was performed using two mitochondrial targeted cationic fluorescent dyes, MitoTracker Green FM (MTG-FM) and . The extracellular flux (XF) assay described here is a plate-based method in which galactose-conditioned HepG2 cells were acutely exposed to test compounds, then real-time changes in the oxygen consumption rate and extracellular acidification rate were simultaneously measured using a Seahorse Bioscience XF-96 analyzer. Associate professor at @UQAM. FCCP Titration in the Presence of Oligomycin in RAW 264.7 Macrophage Cells RAW 264.7 macrophage cells were seeded into XF e 96 microplates (Seahorse Bioscience) at a density of 8.0×10 4 cells/well. For detailed FCCP titration experiments, see supplemental Fig. Thus, for each new cell line tested, careful titration of the uncoupler is always good practice to minimize toxicity problems. in complete media can be assayed in the Seahorse EFA. Analyzing Microgram Quantities of Isolated Mitochondria in the Agilent Seahorse XFe/XF96 Analyzer Application Note Introduction Enhanced appreciation for the role of altered mitochondrial function in tumorigenesis, aging, neurodegenerative, metabolic, and cardiovascular diseases has stimulated the development of a variety of new approaches for assessing mitochondrial function and dysfunction. A key platform for testing candidate chemotherapeutic compounds is the intrahepatic orthotopic xenograft (IOX) model in rodents. Non-phosphorylating respiration (O) represents the residual respiration in the presence of oligomycin, whereas the maximal uncoupled stimulated respiration (F) was determined by titration of FCCP. Because RAW 264.7 cells are semi-adherent, cells were added to each well and the microplate was then centrifuged at 300 x g for two minutes to settle . ( a ) Oxygen consumption rates were measured in both Opa1 −/− and Opa1 +/+ MEFs with the Seahorse XFe96 extracellular flux analyzer. Eugenia Trushina. Frontiers in Neurology, 2012. Preparation of KHB buffer . 1 F). For oxygen consumption assay by Seahorse XF24 flux analyzer, 50,000 control osteosarcoma cybrids cells in 200-μ L medium/well was plated on 24-well plates and incubated overnight. 10.5 FCCP (0.004 mg): Prepare a 250 µM FCCP stock solution (100X) by dissolving vial contents in 60 µL of DMSO. Seahorse Bioscience recommends freshly preparing, testing and optimizing your solution of . III.4. only. For my rat heart cells,. 9. After preincubation, uptakes of [14 C]salicylic acid (6 μM;A) and l-[14 C]lactic acid (3 μM;B) were measured at 37°C by incubating BeWo cells in uptake buffer for 30 s in the absence ( ) and presence ( ) of FCCP . Load the cartridge in each port as outlined below. The rate of oxygen consumption was measured using a Seahorse XF24 analyzer in the HepG2 cells before and after treatment with various concentrations of (A) FCCP and chlorfenapyr, and (B) rotenone and pinacyanol. Final concentration of FCCP is 1uM (HME-shV, HME-shCCN6), 0.25uM (MDA-MB-231-Flag) and 0.125uM (MDA-MB-231-CCN6). FCCP titration was performed to determine the maximal concentration that can induce the maximal OCR in each cell type. 5B, Method 2b) 1b. Aliquot FCCP stock solution so that you have enough volume to perform the desired number of assays (recommendation: 20 µL to cover 20 tests). Blood-based bioenergetic profiling has promising applications as a minimally invasive biomarker of systemic bioenergetic capacity. Test Kit (Seahorse Bioscience), were added sequentially. In general, FCCP is effective in a very narrow range of concentrations, which can be limiting in certain experimental settings, especially if this concentration is not high enough to dissipate the proton gradient. 3 extracellular flux analyzer (Seahorse Bioscience, Billerica, MA). Because RAW 264.7 cells are semi-adherent, cells were added to each well and the microplate was then centrifuged at 300 x g for two minutes to settle . The working concentrations for each inhibitor were as follows: oligomycin 1 µmol/L, FCCP 4 µmol/L, rotenone 0.5 µmol/L, and antimycin A 0.5 µmol/L. . Relative value of spare respiratory capacity was determined by this calculation: Maximal OCR / (Basal OCR *100); lower part Schematic 4(a), left]. These assays function as experimental platforms for understanding the basic mechanisms of hematopoiesis, angiogenesis . Changes in mitochondrial dynamics and function contribute to progression of multiple neurodegenerative diseases including peripheral neuropathies. or FCCP is the lowest concentration that maximally decreases or increases OCR, respectively. Calibration buffer (Seahorse Bioscience) 1.6. Prepare 150 µM Rotenone: Dissolve 3.94 mg of Rotenone in 10 mL of pure ethanol to prepare 1 mM stock, vortex until fully dissolved. Obtain a hydrated cartridge from the non-CO 2 incubator. Prepare serial dilutions of FCCP in assay medium, starting with Tube a as detailed below: c. Pipette 300 μL of the rotenone/antimycin A stock into a 2700 μL aliquot of assay medium: 9. Thus, we recommend performing a six-point FCCP titration (0.125, 0.25, 0.5, 1.0, 1.5, 2.0) along the x-axis of the plate as part of your first MST experiment. Store at - 20°C. For Seahorse metabolic profiling, . FCCP is the most commonly used uncoupling agent, which can be cytotoxic in high concentration. The spare respiratory capacity is determined after oligomycin and FCCP injections. Preparation of KHB buffer . Because RAW 264.7 cells are semi-adherent, cells were added to each well and the microplate was then centrifuged at 300×g for two minutes to . The FCCP titration (2-12.5 µM) of ZAP-70 + CLL cells demonstrated that they were more sensitive to FCCP compared to ZAP-70 − CLL cells or B lymphocytes, achieving a maximal respiratory capacity at 5.32 ± 1.91 µM, as opposed to 8.50 ± 2.42 µM in B lymphocytes and 7.18 ± 2.43 µM FCCP in ZAP70 − CLL cells (Figure 4H), demonstrating . with SeaHorse assay media, and the plate was incubated at 37°C in an incubator without CO 2 for 45min immedi-ately before the assay to allow medium temperature and pH to reach equilibrium. The initial titration of carbonyl cyanide p-(triﬂuoromethoxy) phenylhydrazone (FCCP) was performed with 2µM FCCP for all assays. The OCR was evaluated with the following injection protocol: oligomycin (2 μg/mL), FCCP1 (0.25 μM) and FCCP2 (1.5 μM in this case) and antimycin A (2 μg/mL). Lactate and glycogen . Carefully, without agitation of the sample material at the bottom of the Seahorse sample plate, remove the MAS buffer from the plate until approximately 80 µl of liquid remains in . Maximal stimulation of OCR was achieved for concentrations between 1.85 and 2.50 μM of FCCP. Because RAW 264.7 cells are semi-adherent, cells were added to each well and the microplate was then centrifuged at 300×g for two minutes to . Maximal stimulation of OCR was achieved for concentrations between 1.85 and 2.50 μM of FCCP. (D) K m of FCCP calculated from FCCP titration (cell lines n = 12, PBMC n = 31). Secondly, FCCP, the uncoupling agent injected, should be optimized for the concentration providing the maximal respiratory effect. 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