Anti cd4 fitc cd25 apc cocktail

Collected MSCs were incubated for 30 min at room temperature with the following specific antibodies: PE mouse anti-human CD29, FITC rat anti-human CD44, FITC mouse anti-human CD105, FITC rat anti-human CD45, APC mouse anti-human CD34, and PE mouse anti-human HLA-DR (all from BD Pharmingen). As a control, the cells were stained with the appropriate isotype antibodies. At the end of co-culture, the CD4⁺ T cell apoptosis was analyzed by using an Annexin V-PE Apoptosis Detection Kit I (BD Biosciences) according to the manufacturer’s instructions. To detect Treg cells, a Human Regulatory T Cell Staining Kit (eBioscience) containing an anti-CD4-FITC/CD25-APC cocktail and anti-Foxp3-PE was used according to the manufacturer’s instruction. In addition, we used a Human Th1/Th2/Th17 Phenotyping kit (BD Pharmingen) to analyze the T helper cell subsets. All samples were analyzed using a BD Biosciences Influx cell sorter.

MSCs were trypsinized for identification of a number of surface markers. The antibodies for surface markers were anti-CD29-PE, anti-CD34-APC, anti-CD44-FITC, anti-CD45-FITC, anti-CD90-PE, anti-CD105-FITC, and anti-HLA-DR-PE (all from BD Pharmingen). PBMCs were harvested through centrifugation at the end of coculture. For proliferation analysis of CD4+ T cells, CFSE-incubated PBMCs were marked with anti-CD4-PE (BD Pharmingen), cells left-shifting into the square gate were recognized as the proliferating cells, and the cell count percentage within this gate represented the proliferation rate. For Th17 analysis, anti-CD4-FITC, anti-CCR4-PerCP-Cy5.5, and anti-CCR6-APC (all from BD Pharmingen) were used. The human regulatory T cell staining kit (eBioscience) containing anti-CD4-FITC/CD25-APC cocktail and anti-Foxp3-PE was used in Treg analysis according to the manufacturer's instruction. Cells were measured in a flow cytometry system (BD FACSVerse).