Comprehensive assessment of chemokine expression profiles by flow cytometry
Jens Eberlein, … , Hugo R. Rosen, Dirk Homann
Jens Eberlein, … , Hugo R. Rosen, Dirk Homann
Published February 8, 2010
Citation Information: J Clin Invest. 2010;120(3):907-923. https://doi.org/10.1172/JCI40645.
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Technical Advance Immunology

Comprehensive assessment of chemokine expression profiles by flow cytometry

Abstract

The chemokines are a large family of mainly secreted molecules involved in the regulation of numerous physiological and pathophysiological processes. Despite many years of investigation, the precise cellular sources of most chemokines have remained incompletely defined as a consequence of the limited availability of suitable reagents to visualize the expression of chemokine proteins at the single-cell level. Here, we developed a simple flow cytometry–based assay using commercially available chemokine-specific antibodies for efficient cell-associated detection of 37 of 39 murine chemokines. To demonstrate the utility of this methodology, we used it to reevaluate the nature of homeostatic chemokines in the hematopoietic compartment, to delineate the complete chemokine profiles of NK cells and B cells in response to major polyclonal stimuli, and to assess the chemokine response of DCs to bacterial infection. The versatility of this analytical methodology was further demonstrated by its application to selected human chemokines and should greatly facilitate any future investigation into chemokine biology at large.

Authors

Jens Eberlein, Tom T. Nguyen, Francisco Victorino, Lucy Golden-Mason, Hugo R. Rosen, Dirk Homann

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Figure 1

Validation of chemokine-specific pAb use for FC.

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Validation of chemokine-specific pAb use for FC. HEK 293T cells were tra...
HEK 293T cells were transfected (Tx) with individual pIRES2-AcGFP1 vectors containing Il15 (negative control) or 36 distinct murine chemokines and cultured for 18 hours. BFA was added for the final 14 hours to limit chemokine secretion, and cells were analyzed for expression of corresponding chemokine proteins by FC as detailed in Methods. All histograms are gated on GFP+ HEK cells comparing Il15 transfectants (gray solid) and respective chemokine transfectants (black tracing) stained with the same chemokine-specific pAb. To reduce proteasomal degradation, Cxcl14-transfected HEK cells were cultured in the presence of 10 μM of the protease inhibitor MG-132 and stained with anti-hCXCL14 mAb clone 131120 or with an anti-hCXCL14 pAb (inset).