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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

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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 5

Chemokine profiles of murine and human NK cells.

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Chemokine profiles of murine and human NK cells.
(A) Splenocytes obtaine...
(A) Splenocytes obtained from B6 mice (or Balb/c mice for CXCL11 analyses) were cultured for 5 hours with PMA/ionomycin and BFA and processed for chemokine FC. Plots are gated on NK1.1+CD3ε– (B6) or DX5+CD3ε– (Balb/c) cells; numbers within plots indicate mean percent IFN-γ+ and/or chemokine-positive cells in the respective quadrants. (B) Chemokine expression by human NK cells (CD56+CD3–) was determined directly ex vivo (constitutive), after 5 hours of culture in the presence of BFA (spontaneous) or stimulation with PMA/ionomycin plus BFA (induced). Vertical markers were set according to goat IgG or isotype control stains. The fraction of chemokine+ NK cells obtained from 4 healthy volunteers is shown below (representative data from 3–5 independent experiments). (C) Blood-borne murine NK cells (NK1.1+CD3ε–) were analyzed for constitutive chemokine expression (black tracings). Different controls (gray solid) are featured in the individual plots (CCL3 control, CCL3 stains of Ccl3–/– NK cells; CCL4 and XCL1 controls, goat IgG stains; CCL5 controls, CCL5 stains of Ccl5–/– and Ccl5+/– NK cells, the latter shown by dotted tracing). The CCL5 expression level (GMFI) of Ccl5+/– NK cells were slightly, but significantly, lower than those of wild-type NK cells (P = 0.01). (D) Constitutive, spontaneous, and cytokine-induced (100 ng/ml IL-2 or IL-15) CCL3/4/5 and XCL1 expression by splenic NK cells was determined in conjunction with IFN-γ; numbers denote average percentages of IFN-γ+ and/or chemokine-positive cells in corresponding quadrants. All data obtained for murine NK cells are representative for multiple independent experiments in 2–3 mice each.

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ISSN: 0021-9738 (print), 1558-8238 (online)

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