% IMPORTANT: The following is UTF-8 encoded.  This means that in the presence
% of non-ASCII characters, it will not work with BibTeX 0.99 or older.
% Instead, you should use an up-to-date BibTeX implementation like “bibtex8” or
% “biber”.

@ARTICLE{Bode:167779,
      author       = {K. Bode$^*$ and C. Link$^*$ and P. H. Krammer$^*$ and H.
                      Weyd$^*$},
      title        = {{F}low-cytometric {D}etection of {L}ow-level {R}eactive
                      {O}xygen {S}pecies in {C}ell {L}ines and {P}rimary {I}mmune
                      {C}ells.},
      journal      = {Bio-protocol},
      volume       = {10},
      number       = {17},
      issn         = {2331-8325},
      address      = {Sunnyvale, CA},
      publisher    = {bio-protocol.org},
      reportid     = {DKFZ-2021-00541},
      pages        = {e3737},
      year         = {2020},
      note         = {#EA:D030#LA:D030#},
      abstract     = {Depending 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.},
      keywords     = {Bone Marrow-derived Dendritic Cells (BMDCs) (Other) /
                      Jurkat T cells (Other) / Mono Mac 6 (MM6) cells (Other) /
                      NADPH-oxidase 2 (NOX-2) (Other) / Reactive Oxygen Species
                      (ROS) (Other)},
      cin          = {D030},
      ddc          = {570},
      cid          = {I:(DE-He78)D030-20160331},
      pnm          = {314 - Tumor immunology (POF3-314)},
      pid          = {G:(DE-HGF)POF3-314},
      typ          = {PUB:(DE-HGF)16},
      pubmed       = {pmid:33659398},
      pmc          = {pmc:PMC7842666},
      doi          = {10.21769/BioProtoc.3737},
      url          = {https://inrepo02.dkfz.de/record/167779},
}