Detection of nuclear FoxP3 expression in CD4+iTregs

February 03, 2015

George Washington University, Washington DC
Surgery
Post-doctoral Fellow

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Company:

BioLegend

Product Name:

FITC anti-human FOXP3 Antibody

Catalog Number:

320106

The role of p53 and its effect on TGF-Beta signaling and subsequent expression of FoxP3 has not yet been identified. We hypothesized that lack of p53 will cause alteration in TGF-Beta signaling by down-regulating expression of TGF-Beta itself and hence CD4+ T cells deficient of p53 (from p53 knock out mice) would not be induced equally to Treg cells.  Expression of FoxP3 was used as a signature marker. Indeed we found that CD4 T cells induced to form iTregs were lacking the expression of the signature Treg marker, FoxP3, which made it evident that lack of p53 inhibits CD4 T cells transformation into Tregs. We believe this is a significant finding in context of immuno-suppression, cancer therapy, and auto-immune diseases with exciting avenues to venture ahead.

Experimental Design and Results Summary

Application

Flow cytometry

Starting Material

CD4+ Tcells

Protocol Overview

Other Reagents needed before starting the protocol: BD Perm/Wash™ buffer (Cat. No. 554723), BioLegend's FOXP3 Fix/Perm buffer set (Cat. No. 421403).  Prepare a 1X working solution of Perm/Wash Buffer by diluting the 10X concentrate with distilled water prior to use.  Prepare fresh Foxp3 Fixation/Permeabilization working solution by diluting Foxp3 Fixation/Permeabilization Concentrate (1 part) with Foxp3 Fixation/Permeabilization Diluent (3 parts).  Working Protocol: Thoroughly resuspend cells and add 100uL per well for microwell plates (or 250 uL for tubes) of Fixation/Permeabilization solution for 20 minutes at 4 degrees C.  NOTE: Cell aggregation can be avoided by vortexing prior to the addition of the Fixation/Permeabilization solutionWash cells two times in 1X BD Perm/Wash™ buffer (e.g., 1 mL/wash for staining in tubes and 250 uL/wash final volume for staining in microwell plates). Centrifuge samples at 300-400xg at room temperature for 5 minutes, then discard the supernatant. NOTE: BD Perm/Wash™ buffer must be maintained in washing steps to keep cells permeabilized. Thoroughly resuspend permeabilized cells in 100 uL BD Perm/Wash™ buffer containing a 1:20 concentration of a FoxP3-FITC conjugated antibody (5ul in 100ul) or appropriate negative control. Incubate at 4 degrees C for 30 minutes in the dark. Centrifuge samples at 300-400xg at room temperature for 5 minutes, then discard the supernatant. Re-suspend cells in appropriate volume of Staining Buffer prior to flow cytometric analysis.

Tips

Concentration of FoxP3 used and time of incubation can be further standardized to obtain best results

Results Summary

Excellent stain for FoxP3 positive Tregs is achievable. We found around 40-45% of Normal CD4+ T cells expressing FoxP3 were transformed to iTregs for 72 hours prior to staining. In contrast a very low percentage of CD4+ T cells lacking p53 expressed FoxP3 (4-5%) depicting perhaps that absence of p53 inhibits the transformation of CD4+T cells to iTregs.

Additional Notes

Cell aggregation can be avoided by vortexing prior to the addition of the Fixation/Permeabilization solution. Perm/Wash buffer must be maintained in washing steps to keep cells permeabilized.

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Summary

The Good

Great staining.  Once standardized, gives consistent and reliable results.

The Bad

Practically nothing

The Bottom Line

Very good Flow cytometric method for analysis of expression of FoxP3, a very important transcription factor in cell siganling and a signature marker for T regulatory cells. Inexpensive and comparatively easy protocol makes this a really good choice.

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