Combined Single-Cell Measurement of Cytokine mRNA and Protein in Immune Cells

  • Julian J. Freen-van Heeren
  • Benoit P. Nicolet
  • Monika C. WolkersEmail author
Part of the Methods in Molecular Biology book series (MIMB, volume 2108)


A key feature of immune cells, such as T cells, is their rapid responsiveness to activation. The response rate of T cells depends on the signal strength, and the type of signals they receive. Studying the underlying mechanisms that define responsiveness, however, is confounded by the fact that immune cells do not uniformly respond to activation. Tools that measure gene products on a single-cell level therefore provide additional insights in T cell biology. Here we describe flow cytometry-based fluorescence in situ hybridization (Flow-FISH), a high-throughput assay that allows for the simultaneous measurement of cytokine mRNA and protein levels of the gene(s) of interest by flow cytometry. We present several possible applications of Flow-FISH in human and murine T cells that—with minor adjustments—should also be applicable for other cell types.

Key words

Flow-FISH Immune cells T cells mRNA Protein Cytokines Flow cytometry Single cell 



We thank A. Guislain, M. Hansen, and B. Popovic for critical reading of this manuscript. This work was supported by the Landsteiner Foundation of Blood Transfusion Research (LSBR) [LSBR Fellowship 1373] and by the Dutch Science Foundation [VIDI Grant 917.14.214] to M.C.W. Julian J. Freen-van Heeren and Benoit P. Nicolet contributed equally to this work.


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Copyright information

© Springer Science+Business Media, LLC, part of Springer Nature 2020

Authors and Affiliations

  • Julian J. Freen-van Heeren
    • 1
  • Benoit P. Nicolet
    • 1
  • Monika C. Wolkers
    • 1
    Email author
  1. 1.Department of HematopoiesisSanquin Research-Amsterdam UMC Landsteiner Laboratory and Oncode InstituteAmsterdamThe Netherlands

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