Human regulatory t cell staining kit 3

FITC-conjugated monoclonal antibodies against CD80 and HLA-DR, as well as PE-conjugated monoclonal antibodies against CD86, CD40, CD45, and HLA-ABC were purchased from BD biosciences (San Jose, CA, USA). PE-conjugated anti-HLA-G antibody was purchased from Abcam (USA). Human Regulatory T Cell Staining Kit #3 (eBiosciences, USA) was used for detecting CD4, CD25, FOXP3 positive cells (Table I).

PBMCs were cultured with MenSCs both cell-cell contact and transwell modes for 72 hr as described in the previous section. Thereafter, PBMCs were harvested from the co-culture and stained using eBioscience ${}^{\mathrm{TM}}$ Human Regulatory T Cell Staining Kit #3 for Treg detection as per manufacturer direction. In brief, the cells were labeled with CD4-FITC and CD25-PE or their isotype-matched control antibodies, washed using stain buffer (PBS with 2% fetal bovine serum [FBS]), fixed, permeabilized, and finally stained intra-cellularly with either PE.Cy5-conjugated anti-human FOXP3 Ab, or its isotype control Ab according to the manufacturer's instructions. Staining was measured using a Partec PAS III flow cytometer (Münster, Germany) and data were analyzed and prepared using the FlowJo software (version 7.6.1.). To measure the percentage of Tregs, CD4+ cells were selected in the lymphocytes gate. Then, CD25+FOXP3+ cells were measured among the CD4+ cells (8).