Phosphoprotein Detection by High-Throughput Flow Cytometry

  • Johannes Landskron
  • Kjetil Taskén
Part of the Methods in Molecular Biology book series (MIMB, volume 1355)


Phospho flow cytometry is a powerful technique for the detection of protein phosphorylation events that, like Western blotting, relies on phospho-epitope-specific antibodies. In contrast to the latter, however, multidimensional and directly quantifiable data is obtained at the single-cell level allowing separate analysis of small cell populations in complex cellular mixtures. Furthermore, up to 30 phospho-specific antibodies or antibodies identifying other posttranslational modifications in combination with cell surface markers can be analyzed in a single experiment. Utilizing a technique called fluorescent cell barcoding that enables combination of up to 64 samples into one tube for multiplex analysis and later data deconvolution, phospho flow cytometry is turned into a medium- to high-throughput technology.

Key words

Flow cytometry Phospho flow Phospho-specific antibody Fluorescent cell barcoding Signaling 



Our research is funded by grants from the Research Council of Norway, Norwegian Cancer Society, the European Commission (7th Framework Programme), South Eastern Norway Regional Health Authority, the K.G. Jebsen Foundation, and Novo Nordic Foundation which has permitted the method development as described here. Johannes Landskron is a Senior Scientist funded by the Jebsen Centre for Cancer Immunotherapy.


  1. 1.
    Krutzik PO, Nolan GP (2003) Intracellular phospho-protein staining techniques for flow cytometry: monitoring single cell signaling events. Cytometry A 55(2):61–70. doi: 10.1002/cyto.a.10072 CrossRefPubMedGoogle Scholar
  2. 2.
    Oberprieler NG, Tasken K (2011) Analysing phosphorylation-based signalling networks by phospho flow cytometry. Cell Signal 23(1):14–18. doi: 10.1016/j.cellsig.2010.07.009 CrossRefPubMedGoogle Scholar
  3. 3.
    Storvold GL, Landskron J, Strozynski M, Arntzen MO, Koehler CJ, Kalland ME, Tasken K, Thiede B (2013) Quantitative profiling of tyrosine phosphorylation revealed changes in the activity of the T cell receptor signaling pathway upon cisplatin-induced apoptosis. J Proteomics 91:344–357. doi: 10.1016/j.jprot.2013.07.019 CrossRefPubMedGoogle Scholar
  4. 4.
    Krutzik PO, Clutter MR, Trejo A, Nolan GP (2011) Fluorescent cell barcoding for multiplex flow cytometry. Current protocols in cytometry/editorial board, J Paul Robinson, managing editor [et al] Chapter 6:Unit 6 31. doi: 10.1002/0471142956.cy0631s55
  5. 5.
    Krutzik PO, Nolan GP (2006) Fluorescent cell barcoding in flow cytometry allows high-throughput drug screening and signaling profiling. Nat Methods 3(5):361–368. doi: 10.1038/nmeth872 CrossRefPubMedGoogle Scholar
  6. 6.
    Kalland ME, Oberprieler NG, Vang T, Tasken K, Torgersen KM (2011) T cell-signaling network analysis reveals distinct differences between CD28 and CD2 costimulation responses in various subsets and in the MAPK pathway between resting and activated regulatory T cells. J Immunol 187(10):5233–5245. doi: 10.4049/jimmunol.1101804 CrossRefPubMedGoogle Scholar

Copyright information

© Springer Science+Business Media New York 2016

Authors and Affiliations

  1. 1.Biotechnology CentreUniversity of OsloOsloNorway
  2. 2.K.G. Jebsen Centre for Cancer ImmunotherapyUniversity of OsloOsloNorway
  3. 3.Centre for Molecular Medicine Norway, Nordic EMBL PartnershipUniversity of OsloOsloNorway
  4. 4.K.G. Jebsen Inflammation Research CentreUniversity of OsloOsloNorway
  5. 5.Department of Infectious DiseasesUniversity of OsloOsloNorway

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