Pe anti human foxp3 staining set

The proportions of different subsets of lymphocytes were analyzed using flow cytometry within 72 h of the cord blood samples being obtained (n = 48), as previously described [29 (link),30 (link)]. In brief, whole blood was incubated for 20 min at 4 °C with the anti-human monoclonal antibodies described in Supplementary Materials Table S1. Red blood cells were then lysed (FACS lysing solution; BD Bioscience, Erembodegem, Belgium) and the remaining cells were washed twice with FACS-buffer. After completion of cell surface staining, the cells were intracellularly stained for FOXP3 using the PE anti-human FOXP3 Staining Set (eBioscience, San Diego, CA, USA). All isotype controls were purchased from BD Bioscience, except for the isotype control for FOXP3 that was purchased from eBioscience. As described in detail previously, the total cell counts of lymphocytes, B cells, and CD4⁺ T cells were determined with the Trucount assay (BD Biosciences) [20 (link)]. Stained samples were analyzed in a FACS Calibur (BD Bioscience) equipped with the CellQuestPro software. Data were examined using the FlowJo software (TreeStar, Ashland, OR, USA). The gating strategy applied has been described previously in detail [29 (link),31 (link)].

Freshly isolated SLN cells, expanded SLN T cells or thawed PBMCs were directly stained with antibodies labeled with either FITC, PE, PE-CY5.5, PerCP-CY5.5 or allophycocyanin and analyzed by flow cytometry at 100,000 or 200,000 events per measurement, as previously described [8 (link), 9 (link)]. Monoclonal antibodies against CD3, CD4, CD8, CD56, CD19, CD25 (BD Biosciences) and latency-associated peptide (LAP) (R&D Systems Inc. Minneapolis, MN) with matching isotype control antibodies were used. Intracellular FoxP3 staining was done using the eBioscience (San Diego, CA) PE-antihuman FoxP3 staining set following the manufacturer’s instructions. CTLA4, IFN-γ, TNF-α, IL-2, IL-4 and IL-5 were stained intracellularly using the Cytofix/Cytoperm Kit with GolgiStop (BD Biosciences) as described [19 (link)]. LAP staining was performed as described [20 (link)]; CD4- and CD25-enriched and CD4- and CD25-expanded T cells were stimulated for 48 h with anti-CD3 and anti-CD28 mAbs (both at 1 μg/ml) in the presence of 10 IU IL-2 after which they were stained according to the published protocol [20 (link)].