SIMPLE AND ROBUST WHOLE BLOOD STAINING PROCEDURE USING PERFIX-NC REAGENTS GENERATES UNPRECEDENTED FOXP3 SIGNAL/NOISE RATIO

Abstract FOXP3 is a nuclear antigen expressed specifically in regulatory T cells (Tregs). Tregs play critical role in the maintenance of the dominant self-tolerance and have been subject of extensive research effort for the last two decades. Today the detection of FOXP3 is laborious, time consuming (3-4 hours) and poorly reproducible. A new commercially available method, named PerFix-nc, allows simultaneous intra- and extra-cellular staining without any wash step. This new kit allows for the reduction of the workflow to 45 min. In this study we aimed at the optimization of the PerFix-nc protocol for the detection of FOXP3. We evaluated various clones from various vendors, optimizing titration and incubation time, and adding some extra washing steps. Signal to noise ratio at least 40% better than the one obtained for the same samples with reference procedure. Improved cell scattered separation compared to reference method was revealed. Purity and recovery were also significantly increased. Furthermore the method was very robust and demonstrated excellent repeatability with respect to percentage of FOXP3-positive cells and signal to noise. Total workflow was reduced from 3-4 hours in reference method to about 95 min. In conclusion, the novel PerFix-nc reagent significantly improves FOXP3 detection in several different ways: it simplifies FOXP3 intracellular staining procedure, generates robust and highly reproducible results, and provides unprecedented signal to noise ratio.