Cell isolation
Inguinal lymph nodes were collected (draining lymph nodes [LNs]) and
used for the experiments. CD4+ cells from draining LNs
were isolated by positive selection using the magnetic activated cell
sorting (MACS) system with anti-CD4 mAb (Miltenyi Biotec, Bergisch
Gladbach, Germany). CD4+GFP+Treg
(Foxp3+Treg) or
CD4+GFP-T cells were isolated with
MoFlo cell sorter (DakoCytomation, Glostrup, Denmark) from MACS-isolated
CD4+ T cells of C57BL/6-Foxp3GFP mice. The isolated
cells were then used forcell culture and flow cytometry.