To the editor – Eur. J. Immunol. 1/2006
It is a promising new tool to investigate regulatory T-cells (Tregs) at the single cell level using intracellular staining with monoclonal antibodies (mAb) directed against the forkhead and winged-helix family transcription factor FOXP3. In their paper, Roncador et al. 1 present a panel of specific mAbs including the commercially available clone hFOXY provided by eBioscience (Cat No 12–5779, eBioscience, San Diego, CA, USA). Although we agree with the authors that studies of FOXP3 expression are necessary because of the lack of concordance between FOXP3 mRNA and protein levels, we question the value of quantifying FOXP3 in human CD4+ cells with varying protocols.
To compare Tween 20 plus DNAse (as used by the authors), Tween 20 alone and the commercially available kit for cell permeabilisation, we isolated peripheral blood mononuclear cells (PBMC), stained them with FITC- and PerCP-conjugated anti-human CD4 mAbs (clone RPA-T4, Becton Dickinson, San Diego, CA, USA) and then permeabilised them according to each of the following protocols: (a) in accordance with the method of Roncador et al. 1 cells were incubated in 1% paraformaldehyde and 0.05% Tween 20 overnight at 4oC and then treated with DNAse at 100 Kunitz/mL (Roche, Grenzach-Wyhlen, Germany) (b) 0.05% Tween 20 was applied for 15 minutes at 37oC after fixation of cells with 1% paraformaldehyde overnight at 4oC without DNAse digestion (c) a commercially available kit was applied according to the manufacturer's instructions (human FOXP3 staining buffers, Cat No 88–5778, eBioscience). IgG1,κ was used as isotype control (eBioscience).
Representative dot blots of four independent experiments are shown in Fig. 1. The percentages of FOXP3 positive cells clearly depends on the applied protocol for permeabilisation of cells: the Tween protocol with DNAse digestion results in the highest percentages of FOXP3+ cells compared with the kit and Tween 20 alone. These experiments emphasize that the staining protocol used is crucial for FOXP3 staining.
Comparison between Tween 20 plus DNAse, Tween 20 alone and the commercially available kit for cell permeabilisation and subsequent staining of FOXP3 with the specific mAb hFOXY and its corresponding isotype.
