Alexa647 foxp3

The following anti-human antibodies were used: CD4-PE/Cy5, CD25-FITC, and Foxp3-Alexa647 from BioLegend (San Diego, CA, USA). Mouse cells were stained with fluorochrome-labeled mAbs to CD4-Pacific Blue, Brilliant Violet 605 and Alexa-700 (RM4-5), CD8-Pacific Blue (53-6.7), CD25-APC (7D4), Ki-67-PE (Ki-67), and Thy1.1-PercP (Ox-7) from BioLegend. Foxp3-APC (FJK-16s), dead cell marker Viability Dye eFluor™ 780, and staining reagents from eBioscience (San Diego, CA, USA) were used according to the manufacturer’s instructions. To analyze expression of surface proteins, the cells were stained with the appropriate antibodies for 20 min at 4°C, washed once with FACS buffer (PBS, 0.1% BSA, and 0.02% NaN₃), and fixed with 2% paraformaldehyde. For intracellular staining of Foxp3 and Ki-67, the cells were first surface stained, permeabilized with Fix/Perm (eBioscience), and stained with Foxp3 and Ki-67 antibodies diluted in Perm/Wash (eBioscience). To calculate absolute Treg numbers, unlabeled microbeads (BD Biosciences, Franklin Lakes, NJ, USA) were added to the stained cells and the following formula was used: absolute Treg numbers = (beads used × Treg events)/beads measured. Acquisition was performed on a BD™ LSR II or FACSCalibur, and data were analyzed using FlowJo software (TreeStar Inc., Ashland, OR, USA).

CD45.2+CD4+ T lymphocytes were sorted from chimeras and differentiated with 10 μg/ml plate coated anti-CD3, 2 μg/ml soluble anti-CD28 and 10 ng/ml TGF-β for five days and analyzed. Thymocytes, lymph node, splenocytes or in vitro differentiated Treg cells were stained with anti-CD4 and anti-CD25 surface markers, then fixed and permeabilized with Fix/Perm buffers (Biolegend) and stained with Alexa647-FoxP3 (Biolegend).