flow cytometry biotinylated polyclonal goat anti human lap  (R&D Systems)

Journal: Virology

Article Title: A TGF-beta mediated regulatory mechanism modulates the T cell immune response to rotavirus in adults but not in children.

doi: 10.1016/j.virol.2009.12.016

Figure Lengend Snippet: Fig. 7. Relative and absolute counts of Treg cells subsets in children with acute GE. Relative (A, C, E) and absolute (B, D, F) numbers of Treg cells subpopulations were determined in PBMC from children with acute GE from groups A+B (circles) and C (diamonds). The cells were stained following protocol 1 (open symbols) or protocol 2 (filled symbols), described in Materials and methods and analyzed as described in Fig. S3A or S3B. Stain 1 and Stain 2 include polyclonal and MAbs against LAP, respectively (described in Materials and methods). Subpopulations of Treg cells were defined as (A, B) CD4+CD25+, (C, D) CD4+CD25+CD127low and (E, F) CD4+CD25+ CD127lowLAP+. Absolute counts were estimated based on lymphocyte numbers from Fig. 4. Similar results to those shown for CD4+CD25+ cells (upper row) were obtained upon analysis of CD4+ CD25hi cells (data not shown). Bars represent median values.

Article Snippet: In the first protocol (stain 1), PBMC were stained with biotinylated goat anti-human LAP (TGF-β1) polyclonal Ab (R&D Systems; Minneapolis, MN) or with control biotinylated goat IgG (R&D Systems) at 4 °C for 30 min. After washing with staining buffer (0.5% bovine serum albumin [Merck, KGaA, Darmstadt, Germany], 0.02% sodium azide [Mallinckrodt Chemicals, Paris, KY] in PBS) cells were stained with anti-CD4 FITC, anti-CD25 APC, anti-CD127 PE MAbs and PerCP conjugated Streptavidin for 30 min at 4 °C.

Techniques: Staining