nTregs were isolated from spleens of BALB/c mice aged 6 to 10 weeks using
antibody-tagged magnetic beads provided in the Mouse CD4+CD25+ Regulatory T Cell
Isolation kit (Miltenyi Biotech Inc, Auburn, CA). To generate alloantigen
induced Tregs, CD4+ T cells purified from BALB/c spleen cells were co-cultured
with B6 splenic DCs, IL-2 and TGF-β1 for 5-6 days, followed by enrichment
of the CD4+CD25+ fraction. The lymphocyte population was based on size and
granularity (R1) gate. In each histogram the red line shows the percentage of
positive cells and MCF values for Foxp3 and CD62L, and the blue line shows
values for isotype controls. Panels on the right show two color analyses for
CD25 and Foxp3, CD25 and CD62L, and for Foxp3 and CD62L. Results shown are
representative of three separate experiments.