Polarization of CD4+ T cells from PBMC's into iTregs identified by FoxP3 expression

October 13, 2016

George Washington University, Washington DC
Surgery
Post-doctoral Fellow

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Company:

BioLegend

Product Name:

PE anti-human FOXP3 Flow Kit

Catalog Number:

320117

Peripheral blood mononuclear cells (PBMC) was isolated from whole blood oof human patient samples. Negative selection was used to isolate CD4+ T cells from PBMC. The CD4+ T cells were then polarized to iTregs for a period of 5 days at the end of which foxP3 expression was analyzed and compared with nTregs (non-polarized). This experiment is useful for analysis of nTregs and iTregs and comparing their ratio.

Experimental Design and Results Summary

Application

Flow Cytometry

Starting Material

PBMC isolated from human blood samples

Protocol Overview

1. Isolate CD4+ T cell by negative selection ( bead/magnet) or by cell sorting.2. Culture CD4+ T cells in iTreg polarizing condition for 96 hours. Keep a set at non polarizing condition as well.3. at the end of 96 hours, do a surface staining of the cells with anti CD4 and antiCD25 antibodies with flourochrome other than PE.4. . Add 1 ml of 1X Biolegend's FOXP3 Fix/Perm solution to each tube, resuspend the cells (gentle vortex) andincubate at room temperature in the dark for 20 minutes, then centrifuge and remove the supernatant. 5. Wash cells in Biolegend 1X Perm buffer.6. stain cells with PE conjugated FoxP3 antibody diluted in 1X perm buffer (1:100) and keep in dark for 30 mins, preferable over ice.7 wash cell in 1X staining buffer and analyze by flow cytometry.

Tips

Always dilute the FoxP3 antibody in perm buffer.

Results Summary

Polarization of CD4+ T cells to iTregs is depicted by the perceent of CD4+T cells expressing FoxP3.

Additional Notes

The fixation/ permeabilization followed by Perm wash is essential to get a proper staining.

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Summary

The Good

Good and Reliable kit to analyze expression of FoxP3 by flow cytometryt

The Bad

Nothing to mention

The Bottom Line

The Biolegend's FoxP3 flow kit is a reliable and fairly simple flow cytometric method for analyzing FoxP3 expression which is a hallmark of T regulatory cells.

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