1 biotin (PK136), T-cell receptor (TCR) γδ (UC7-13D5), TCR-β (H57-597), CD127 Alexa Fluor 647/phycoerythrin (PE) (A7R34), CD25 FITC/APC (PC61.5), Streptavidin efluor 450, CD16/32 PE-Cy7 (93), CD4 PE-Cy7 (GK1.5), CD44 PE-Cy7 (IM7), CD23 (B3B4), CD21 (8D9), CD80 (16-10A1), MHC II

(M5/114.15.2), IgM (11/41), IgD (11-26), CD93 (AA4.1) and CD43 (R2/60). Immature, DN thymocytes were stained with a pool of antibodies recognizing lineage (Lin) markers. The lineage mix contained antibodies to B220, CD3ε, CD8β, CD8α, CD11b, Gr-1, CD11c, NK1.1, TCR-β, and TCR-γ as previously described.[21] The DN thymocytes, after lineage gating, were further characterized into DN1 (CD44+ CD25−), DN2 (CD44+ CD25+). DN3 (CD44− CD25+),

and DN4 (CD44− CD25−) populations.[22] Early T-lineage progenitors (ETPs) after lineage gating, were defined as CD44+ CD25− c-Kithi IL-7R−/lo.[21] KU-57788 concentration Effector/effector memory splenic T cells were defined as CD44hi CD62Llo, and central memory T cells were defined as CD44hi CD62Lhi.[23] Bone marrow B cells were defined based upon previously reported markers.[24, 25] Pre-pro B cells were defined as Erlotinib research buy B220+ CD19− CD43+ IgM−, pro-B cells were defined as B220+ CD19+ CD43+ IgM−, pre-B cells were defined as B220+ CD19+ CD43− IgM−, immature B cells were defined as B220+ CD19+ CD43− IgM+, and mature B cells were defined as B220+ IgM+ IgD+. In the spleen, B-cell subsets were defined as find more described by Allman and Pillai.[26] CD19+ B cells were defined as transitional (T) B-cell subsets; T1: B220+ AA4+ IgMhi CD23−; T2: B220+ AA4+ IgMhi CD23+; T3: B220+ AA4+ IgMlo CD23+ or marginal zone (MZ) B-cell subsets; MZ: B220+ AA4− IgMhi CD21hi CD23−; or marginal zone precursor (MZP): B220+ AA4− IgMhi CD21hi CD23+, or follicular (Fol) B-cell subsets were defined as Fol I: B220+ AA4− IgMlo CD21lo IgD+; or Fol II: B220+ AA4− IgMhi CD21lo IgD+. Compensation settings and lineage gates were based upon

single colour controls. Analysis was performed with FlowJo (Tree Star, Inc., Ashland, OR) Intracellular reactive oxygen species were analysed in selected subsets by using the oxidation sensitive dye dichlorodihydrofluorescein diacetate (DCFDA) as previously described.[6] Cells were incubated ex vivo with 2 μm DCFDA at 37° for 15 min, washed and surface stained. As a loading control, parallel samples were incubated with the oxidized control dye fluorescein diacetate (FDA) (0·01 μm) at 37° for 15 min, washed, and surface stained as described above. FACS analysis was performed immediately. DCFDA mean channel fluorescence was normalized to FDA uptake, and the data are shown as the per cent increase in DCFDA fluorescence in cells from Ts65Dn mice over euploid controls ± SEM. Intracellular glutathione levels were measured in progenitor subsets by flow cytometry using monochlorobimane (MCB) essentially as previously described.