Identification of Porcine Regulatory T Cells

Tregs were determined by coexpression of CD4, CD25 and Foxp3. After 3 days co-culture with PBMCs and MoDCs [34 (link)], all cells were harvested and stained with anti-porcine CD25 (AbD Serotec), followed by APC goat anti-mouse IgG (H+L) (Life Technologies) and goat anti-porcine CD4a-FITC (Clone 74-12-4; SouthernBiotech). Foxp3 intracellular staining was performed with anti-rat/mouse Foxp3-PE (Clone FJK-16s; eBioscience), which had cross-reactivity with swine Foxp3 [26 (link)], using the Foxp3 Staining Buffer Set (Staining, Fixation/Permeabilization and Permeabilization Buffers; eBioscience), to obtain the three-color staining CD4^FITC CD25^APC Foxp3^PE. The Tregs frequency was evaluated by flow cytometry (BD FACS Canto II), and data were analyzed using FlowJo version 7.6.1 software.