Pe conjugated anti cd45ro clone uchl 1

Total numbers and phenotypic characterization of T and B cells in the FARMFLORA study were determined by flow cytometry as previously described in detail.^{4 , 5 (link)} For quality assurance, the same buffers, fluorochromes and antibody clones were used for analysis of cells in umbilical cord blood as well as in peripheral blood sampled at 3–5 days, and at 1, 4 and 18 months of age. The following antihuman monoclonal antibodies from BD Biosciences (Erembodegen, Belgium) were used: PerCP-conjugated anti-CD4 (clone SK3) and anti-CD20 (clone L27); allophycocyanin (APC)-conjugated anti-CD25 (clone 2A3) and anti-CD5 (clone UCHT2); phycoerythrin (PE)-conjugated anti-CD45RO (clone UCHL-1); FITC-conjugated anti-CD27 (clone L128) and biotin-conjugated anti-CTLA-4 (clone BN13). The PE-antihuman FOXP3-staining set (clone PCH101) was purchased from eBiosciences (San Diego, CA, USA); and the Cytofix/Cytoperm kit (BD Biosciences) was used for detection of CTLA-4. Samples were run in an FACS-Calibur (BD Biosciences) equipped with CellQuestPro software. All samples were analyzed with FlowJo software (TreeStar, Ashland, OR, USA). Gating strategies for T- and B-cell subsets are demonstrated in Supplementary Figure S1.

The proportions of CD4⁺ T cells that were CD45RO-positive were analyzed by flow cytometry within 72 hours after sampling, as previously reported in detail.^{14–16 (link)} The following antibodies were used: PerCP-conjugated anti-CD4 (clone SK3, BD Bioscience) and PE-conjugated anti-CD45RO (clone UCHL-1, BD Bioscience). Isotype controls were purchased from BD Bioscience. A FACSCalibur (BD Bioscience) equipped with CellQuestPro software was used to examine stained samples and flow cytometry data were analyzed using Flow Jo software (TreeStar, Ashland Oregon).