Intracellular Staining for IL-17 by Flow Cytometry

March 30, 2016

Surgery
George Washington University, Washington DC
Post-doctoral Fellow

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Company:

BioLegend

Product Name:

FITC anti-mouse IL-17A Antibody

Catalog Number:

506908

IL-17–producing CD8+ T (Tc17) cells are important mediators of inflammation and has been proven to have anti-tumor effects. But excess production of IL-17 has been a major cause in driving inflammatory response in several autoimmune diseases. our aim for this experiment was to find differences in production of IL-17(A), when mouse splenocytes were polarized to either Tc1 or Tc17 cells. Tc17 cells has the ability to generate abundance of IL-17 and thus inhibition of Tc17 cells can limit IL-17 production and help reduce severe effects in autoimmune diseases.

Experimental Design and Results Summary

Applications

Flow Cytometry

Sample

Mouse spleen cells

Primary Incubation

Mouse splenocytes were activated by anti-CD3/anti-CD28 for 96 hours, in polarizing conditions towards Tc1/Th1 or Tc17/Th17, followed by restimulation with PMA/Ionomycin in presence of brefeldin-A (golgi-stop). Cells were washed after 96 hour incubation and re-suspended in 1X FACS buffer. Staining of cell surface receptors were done in the first step. Cells were incubated for 30 mins in APC conjugated anti-CD8 (mouse) in the dilution of 1:100, in FACS buffer. Cells were washed twice after staining and the next step of intracellular staining was carried out.

Blocking Agent

Fixation of cells was done in BD fix/perm buffer for 15 mins. in ice. Cells were then washed with BD perm/wash buffer.

Secondary Incubation

Cells were incubated for 30 mins in IL-17A antibody(FITC) and IFN-g (PE) diluted in perm/wash buffer with dilution of 1:100 (kept on ice)

Tertiary Incubation

NA

Detection

Detection of intracellular ILK-17 was done by Flow cytometry. Briefly, cells were first gated on CD8+ cells in the APC channel. Next histograms were made for IL-17 and IFN-g for PE vs FITC on the gated CD8+ cells.

Results Summary

Results indicated that Tc17 polarized cells had a higher production of IL-17 compared to TC1 (with higher IFN-g), suggesting that IL-17 is the key mediator for inflammatory response in TC 17 cells.

Additional Notes

Always perform staining for extracellular receptors first when staining for intracellular cytokines

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Summary

The Good

Excellent intracellular cytokine detection method

The Bad

Nothing to point out.

The Bottom Line

Intracellular staining of cytokines (IL-17 & IFN-g) for this experiment using flow cytometry is a convenient and reliable method to analyze cytokine production by a specific subset of cells.

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