Expression Of CCR6 By In-Vitro Differentiated Th17 Cells

October 21, 2016

Surgery
George Washington University, Washington DC
Post-doctoral Fellow

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Company:

BioLegend

Product Name:

PE anti-human CD196 (CCR6) Antibody

Catalog Number:

353410

CD196 or CCR6 is a g-protein coupled chemokine receptor which is preferentially expressed by memory T cell and immature dendritic cells. CCR6 surfaace expression has been reported to be a marker for Th17 differentiated cells. CCR6 also has a role in migration and homing of these proinflammatory cell subsets. PBMC's isolated from human blood samples were differentiated in-vitro to Th17 cells and expression of CCR6 was measured on day 5 of culture.

Experimental Design and Results Summary

Applications

Flow Cytometry

Sample

In-vitro differentiated Th17 cells from human PBMCs.

Primary Incubation

Cells were stained with 100 ul of staining buffer for each sample containing either PE conjugated CCR6 antibody or the corresponding isotype in 1:100 concentration and kept in dark for 25-30 mins. Cells were washed twice staining buffer and resuspended in 500ul of staining buffer for analysis.

Blocking Agent

NA

Secondary Incubation

NA

Tertiary Incubation

NA

Detection

Flow cytometric detection of PE conjugated anti-CCR6 human antibody

Results Summary

Th17 cells were polarized using 0.5 μg/mL anti-CD28, 1 μg/mL anti-IFN-γ, 1 μg/mL anti-IL-2, and 1 μg/mL anti-IL-4 antibodies, 20 ng/mL human IL-6, and 1 ng/mL TGF-β1 in RPMI1640 supplemented with 10 % FBS. On day 5 of culture cells were washed and stained with PE conjugated anti-CCR6 human antibody. Th17 differentiated (CD4+ gated) cells showed upregulation of CCR6 expression detected by a high percentage of CCR6+ cells.

Additional Notes

Other surface markers (CD4; CD8; CXCR3) and intracellular cytokines (IL-17) were also stained for the same set of cells in the same panel. Here we discuss the expression of CCR6 only.

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Summary

The Good

Simple method of detection of chemokine receptor from live cells using flow cytometry.

The Bad

Did not find anything to mentioon

The Bottom Line

Expression/ upregulation of surface receptors are important determinants of cellular function and activation. Detection of the chemokine receptor CCR6 is important to understand the inflammatory, migratory and homing nature of the cell and this method using PE conjugated anti-CCR6 human antibody, provides a simple illustration of identification of CCR6 surface expression using flow cytometry.

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