000167779 001__ 167779
000167779 005__ 20240229123236.0
000167779 0247_ $$2doi$$a10.21769/BioProtoc.3737
000167779 0247_ $$2pmid$$apmid:33659398
000167779 0247_ $$2pmc$$apmc:PMC7842666
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000167779 037__ $$aDKFZ-2021-00541
000167779 041__ $$aEnglish
000167779 082__ $$a570
000167779 1001_ $$0P:(DE-He78)322352fbba1747192994bd6eff973bd8$$aBode, Kevin$$b0$$eFirst author$$udkfz
000167779 245__ $$aFlow-cytometric Detection of Low-level Reactive Oxygen Species in Cell Lines and Primary Immune Cells.
000167779 260__ $$aSunnyvale, CA$$bbio-protocol.org$$c2020
000167779 3367_ $$2DRIVER$$aarticle
000167779 3367_ $$2DataCite$$aOutput Types/Journal article
000167779 3367_ $$0PUB:(DE-HGF)16$$2PUB:(DE-HGF)$$aJournal Article$$bjournal$$mjournal$$s1615279623_28694
000167779 3367_ $$2BibTeX$$aARTICLE
000167779 3367_ $$2ORCID$$aJOURNAL_ARTICLE
000167779 3367_ $$00$$2EndNote$$aJournal Article
000167779 500__ $$a#EA:D030#LA:D030#
000167779 520__ $$aDepending on its concentration and cellular origin the production of reactive oxygen species (ROS) in the organism serves a variety of functions. While high concentrations during an oxidative burst are used to fight pathogens, low to moderate amounts of ROS act as signaling molecules important for several physiological processes such as regulation of immune responses. The ROS-sensitive dye 2',7'-dichlorodihydrofluorescein diacetate (H2DCFDA) is an inexpensive and well-established tool for measuring intracellular ROS levels. However, it needs to be carefully controlled to be able to draw firm conclusions on the nature of ROS species produced and the cellular source of ROS generation such as the enzyme complex NADPH-oxidase 2 (NOX-2). In this protocol, a robust method to determine low intracellular ROS production using H2DCFDA was validated by several ROS-specific as well as NOX-2-specific inhibitors. Cells were treated with inhibitors or control substances prior to treatment with the ROS-inducer of interest. H2DCFDA was added only for the last 30 min of the treatment schedule. To terminate its conversion, we used a ROS-specific inhibitor until analysis by flow cytometry within the FITC-channel (Ex: 488 nm/Em: 519 nm). In summary, this protocol allows the detection of signaling-relevant intracellular ROS production in cell lines and primary immune cells (e.g., Mono Mac 6 cells and Bone marrow-derived dendritic cells, respectively). Using this method in combination with specific inhibitors, we were able to validate even exceptionally low amounts of ROS produced by NOX-2 and relevant for immune-regulatory signaling.
000167779 536__ $$0G:(DE-HGF)POF3-314$$a314 - Tumor immunology (POF3-314)$$cPOF3-314$$fPOF III$$x0
000167779 588__ $$aDataset connected to CrossRef, PubMed,
000167779 650_7 $$2Other$$aBone Marrow-derived Dendritic Cells (BMDCs)
000167779 650_7 $$2Other$$aJurkat T cells
000167779 650_7 $$2Other$$aMono Mac 6 (MM6) cells
000167779 650_7 $$2Other$$aNADPH-oxidase 2 (NOX-2)
000167779 650_7 $$2Other$$aReactive Oxygen Species (ROS)
000167779 7001_ $$0P:(DE-He78)816f10fe44243f27b2b9a15d421dd186$$aLink, Corinna$$b1$$udkfz
000167779 7001_ $$0P:(DE-He78)92492c6eae05ee58973fc142c9201e3d$$aKrammer, Peter H$$b2$$udkfz
000167779 7001_ $$0P:(DE-He78)4dca42511637e2f9d25519745fa3c697$$aWeyd, Heiko$$b3$$eLast author$$udkfz
000167779 773__ $$0PERI:(DE-600)2833269-6$$a10.21769/BioProtoc.3737$$gVol. 10, no. 17$$n17$$pe3737$$tBio-protocol$$v10$$x2331-8325$$y2020
000167779 909CO $$ooai:inrepo02.dkfz.de:167779$$pVDB
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000167779 9101_ $$0I:(DE-588b)2036810-0$$6P:(DE-He78)816f10fe44243f27b2b9a15d421dd186$$aDeutsches Krebsforschungszentrum$$b1$$kDKFZ
000167779 9101_ $$0I:(DE-588b)2036810-0$$6P:(DE-He78)92492c6eae05ee58973fc142c9201e3d$$aDeutsches Krebsforschungszentrum$$b2$$kDKFZ
000167779 9101_ $$0I:(DE-588b)2036810-0$$6P:(DE-He78)4dca42511637e2f9d25519745fa3c697$$aDeutsches Krebsforschungszentrum$$b3$$kDKFZ
000167779 9131_ $$0G:(DE-HGF)POF3-314$$1G:(DE-HGF)POF3-310$$2G:(DE-HGF)POF3-300$$3G:(DE-HGF)POF3$$4G:(DE-HGF)POF$$aDE-HGF$$bGesundheit$$lKrebsforschung$$vTumor immunology$$x0
000167779 9132_ $$0G:(DE-HGF)POF4-314$$1G:(DE-HGF)POF4-310$$2G:(DE-HGF)POF4-300$$3G:(DE-HGF)POF4$$4G:(DE-HGF)POF$$aDE-HGF$$bGesundheit$$lKrebsforschung$$vImmunologie und Krebs$$x0
000167779 9141_ $$y2020
000167779 915__ $$0StatID:(DE-HGF)0300$$2StatID$$aDBCoverage$$bMedline
000167779 9201_ $$0I:(DE-He78)D030-20160331$$kD030$$lImmungenetik$$x0
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000167779 980__ $$aI:(DE-He78)D030-20160331
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