Cd4 pc7

Cells were stained using a standard method described by the manufacturer (eBioscience, United States). In brief, a single cell suspension was incubated with Fc Block (eBioscience) on ice for 20 min. Cells were stained with CD4-PC7, CD4-PE-Cy7, Foxp3-PE, CD45RB-APC (eBioscience) and CD25-PE (Miltenyi Biotec, United States) in the dark for 30 min at 4 °C. Viability staining was performed using eFluor450 (eBioscience). For intracellular staining, cells previously stained for membrane proteins were fixed and permeabilized using a Fix/Perm kit (eBioscience). Cells were visualized using the BD LSRII analyzer (BD Biosciences, United States) and data were analyzed using FlowJo software, version 9.6 (Tree Star, United States).

The T cell-dependent model of colitis was adapted from Ostanin et al[5 (link)]. Briefly, single cell suspensions of SMNC were enriched for CD4⁺ T cells using the Negative T cell Isolation kit (Miltenyi Biotec). The CD4⁺ enriched fraction was stained with CD4-PC7, CD45RB-APC (eBioscience) and CD25-PE (Miltenyi Biotec) and sorted on a BD FACS Aria II cell sorter (BD Biosciences, United States) into CD4⁺CD25⁺CD45RB^low Treg and CD4⁺CD25^-CD45RB^high Teff. CD45RB^low Treg and CD45RB^high Teff were adjusted to a concentration of 2 × 10⁶ cells/mL and 1 × 10⁷ cells/mL respectively in Hank’s balanced salt solution (HBSS). Sham treated mice were injected i.v. with 100 μL of HBSS; the “no Treg” (Teff only) group was infused with 0.5 × 10⁶ CD45RB^highfgl2^+/+ Teff cells; the fgl2^+/+ and fgl2^Tg Treg-treated groups were infused with 0.5 × 10⁶ CD45RB^highfgl2^+/+ Teff cells and 0.1 × 10⁶ CD45RB^low Treg isolated from fgl2^+/+ or fgl2^Tg mice. Mice were weighed weekly and were sacrificed at 14 wk post cell transfer or when they had lost 20% of body weight.