Anti pe antibody conjugated microbeads

CD4+CD25− cells and CD4+CD25+ cells (hereinafter T_regs) were isolated from the PBMC population with the help of immunomagnetic beads as previously described^{14 (link)}. CD4+CD25− cells were further analyzed and purified using CD7 expression. In short, CD4+CD25− cells were treated with anti-CD7 (PE-conjugated) for 30 min followed by incubation with anti-PE antibody-conjugated microbeads (Miltenyi Biotec, Bergisch-Gladbach, Germany) for 15 min (4 °C). Then, the resulting CD4+CD25−CD7+ cells and CD4+CD25−CD7− cells were purified by positive and negative selection, respectively, with MS separation columns (Miltenyi Biotec). CD4+CD25−CD7+ cells were further analyzed and sorted according to their levels of CD43 and CD45 expression (i.e., CD43_low, CD43_high, CD45_low, and CD45_high).
Similarly, CD4+CD25+ cells were treated with anti-Gal1 (PE-conjugated) for 30 min followed by incubation with anti-PE antibody-conjugated microbeads (Miltenyi Biotec) for 15 min (4 °C). Then, the resulting CD4+CD25+Gal1+ cells and CD4+CD25+Gal1− cells were purified by positive and negative selection, respectively, with MS separation columns (Miltenyi Biotec) and were further analyzed and sorted according to their levels of CD25 expression (i.e., CD25_low, CD25_med, and CD25_high).