The importance of CD4+CD25+ Regulatory T (Treg) cells is being recognized in clinical aspects of various fields such as tumor and microbial immunities, transplantation and allergy. Intensive research has been employed to determine exclusive markers for Treg cells. So far, the forkhead transcription factor (FoxP3) is widely accepted as the most specific and reliable marker for detection of natural CD4+CD25+ Treg cells. Thus flow cytometric analysis of FoxP3 protein expression gives the best chance for identifying these cells in a real-time assay at the single-cell level. However, identification of these important regulatory T cells using FoxP3 intracellular staining is usually difficult because of the lack of specific antibodies for FoxP3.
In our laboratory, we were looking for reliable reagents for Treg identification so that we could monitor these cells in a number of different experimental settings. So when we found the Human Regulatory T Cell Staining Kit from eBioscience, we were excited and enthusiastic to try it. This kit contains all the reagents required to stain the natural Treg cells including CD4, CD25 and FoxP3 monoclonal antibodies, IgG2a isotype control, buffers, fixation and permeabilization solutions and normal rat serum. The protocol has been optimized for staining human Treg cells in peripheral blood mononuclear cells (PBMC).
The procedure is quite easy and takes three to four hours to complete. Prepared cells can be first stained for extracellular CD4 and CD25 markers using CD4/25 cocktail (a cocktail of anti-human CD4-FITC and anti-human CD25-APC). Following fixation and permeabilization (using the fix/perm buffers included in the kit), the cells are washed and blocked for non-specific binding sites using normal rat serum. Then anti-human FoxP3-PE or rat IgG2a-PE isotype negative control are then added for 30 minutes before washing twice and flow cytometric analysis. A two-laser cytometer, such as the Becton Dickinson FACSCalibur, which can detect up to four different fluorescence intensities is required for sample acquisition. Analysis can be performed by gating based on CD4 expression and applying this gate on a dot plot of CD25 against FoxP3 to calculate the percentage of CD4+CD25+FoxP3+ T cells.
In our experience, the Human Regulatory T Cell Staining Kit from eBioscience is a reliable and straightforward tool for detection of CD4+CD25+ Treg cells expressing FoxP3. The protocol recommends using 1 x 10^6 PBMCs but it can be adjusted for staining other cells (e.g. expanded effectors or TILs). One kit is sufficient for staining 25 samples. Overall, I am very satisfied with this kit and absolutely recommend it especially for anyone facing problems with intracellular FoxP3 staining.
Eyad Elkord, Ph.D.
Post-Doctoral Fellow
Paterson Institute for Cancer Research
The University of Manchester, UK