cd205 alexa 647  (Bio-Rad)

Journal: The Journal of Experimental Medicine

Article Title: Lung-resident tissue macrophages generate Foxp3 + regulatory T cells and promote airway tolerance

doi: 10.1084/jem.20121849

Figure Lengend Snippet: Phenotypic characterization of lung-resident MØs and DCs in naive mice. Lungs from naive, unmanipulated, unsensitized mice were lavaged and perfused, and lung tissue was digested to make single-cell suspensions. (A) Cells were stained for CD45 and CD11c and further gated (R1) as CD45 + CD11c + for analysis of AF. CD11c + CD45 + cells contained AF-high (R2) and AF-low (R3) cells, which were further characterized as MHC II lo , F4/80 + , Siglec F + versus MHC II hi , F4/80 − , Siglec F − , respectively. (B) Lung MØs (R2) and DCs (R3) gated as in A were stained for CD11b, CD205, CD24, and CD68. (C) Lung MØs (CD11c + , AF hi , Siglec F + , MHC II lo , F4/80 + ) and lung DCs (CD11c + , AF lo , Siglec F − , MHC II hi , F4/80 − ) were sorted from enriched Siglec F + or CD11c + cells as described in Materials and methods and visually assessed in cytospins. Bar, 50 µm. The purity of these two populations after sorting was typically >99% as measured by flow cytometry. Data are representative of two (B and C) or three (A) independent experiments.

Article Snippet: The following antibodies were purchased from BD: anti–CD4-allophycocyanin, anti–CD4-PerCp, anti–CD4-FITC, anti–CD11c-FITC or -allophycocyanin, anti–CD11b-FITC or -allophycocyanin, anti-CD24–Alexa Fluor 647, anti–CD45-PerCp, anti–CD45.2-FITC or -allophycocyanin, anti–CD45.1-FITC or -allophycocyanin, anti–CD62L-FITC, anti-CD68–Alexa Fluor 647, anti-CD205–Alexa Fluor 647, anti–I-A/I-E–PE, anti–Vα2-PE, anti–Vβ5-FITC, and anti–Siglec F–PE.

Techniques: Staining, Flow Cytometry

Journal: The Journal of Experimental Medicine

Article Title: Lung-resident tissue macrophages generate Foxp3 + regulatory T cells and promote airway tolerance

doi: 10.1084/jem.20121849

Figure Lengend Snippet: Lung tissue MØs induce iT reg cell differentiation in vitro. (A and B) Foxp3 − OT-II CD4 T cells were stimulated with purified lung tissue MØs or DCs at a 1:25 APC/T ratio in the presence of OVA peptide for 5 d. (A, top) Foxp3 intracellular staining before and after APC culture. (bottom) Mean percentage (left) and mean number (right) ± SD of Foxp3 + OT-II cells generated by lung MØs and DCs from three to four independent experiments. (B, top) Intracellular IFN-γ and IL-10 expression before and after APC culture. (bottom) Mean percentage ± SD of T cells expressing IFN-γ (left) and mean proliferation (tritiated thymidine incorporation) ± SD of CD4 T cells (right) from three to four independent experiments. (A and B) *, P < 0.01. (C and D) Naive mice were administered OVA-conjugated Alexa Fluor 647 i.n. 24 h later, lung MØs (Alexa Fluor 647 + CD11c + Siglec F + AF hi ), lung DCs (Alexa Fluor 647 + CD11c + Siglec F − AF lo ), and MLN DCs (Alexa Fluor 647 + CD11c + B220 − ) that had taken up OVA were then sorted (see Materials and methods). (C) Representative profile of Alexa Fluor–OVA versus Siglec F expression in gated CD11c + cells for lung MØs and Alexa Fluor–OVA versus CD11c expression in gated Siglec F − cells for lung and MLN DCs before and after sorting. (D) Purified OVA-loaded MØs and DCs were prepared as in A and cultured with Foxp3 − OT-II CD4 T cells at a 1:25 APC/T ratio for 5 d, and induction of Foxp3 + CD4 T cells was analyzed. Data are representative of three independent experiments with APCs purified from groups of 8–10 mice. (E) CFSE-labeled naive OT-II CD4 T cells were stimulated with OVA-pulsed T-depleted splenocytes (APCs) for 4 d in the absence (Teff alone) or presence (Foxp3 + : Teff) of 1:10 or 1:1 ratios of Foxp3 + T cells generated from lung MØ cultures as in A. Data are representative of two experiments.

Article Snippet: The following antibodies were purchased from BD: anti–CD4-allophycocyanin, anti–CD4-PerCp, anti–CD4-FITC, anti–CD11c-FITC or -allophycocyanin, anti–CD11b-FITC or -allophycocyanin, anti-CD24–Alexa Fluor 647, anti–CD45-PerCp, anti–CD45.2-FITC or -allophycocyanin, anti–CD45.1-FITC or -allophycocyanin, anti–CD62L-FITC, anti-CD68–Alexa Fluor 647, anti-CD205–Alexa Fluor 647, anti–I-A/I-E–PE, anti–Vα2-PE, anti–Vβ5-FITC, and anti–Siglec F–PE.

Techniques: Cell Differentiation, In Vitro, Purification, Staining, Generated, Expressing, Cell Culture, Labeling

Journal: The Journal of Experimental Medicine

Article Title: Lung-resident tissue macrophages generate Foxp3 + regulatory T cells and promote airway tolerance

doi: 10.1084/jem.20121849

Figure Lengend Snippet: Activated CD4 T cells primed by MLN DCs become iT reg cells after encounter with lung tissue MØs. WT mice were administered OVA–Alexa Fluor 647 i.n., and after 24 h, OVA-loaded DCs from MLNs were sorted as in . These DCs were cultured with Foxp3 − OT-II T cells for 1 d and assessed for activation markers and induction of Foxp3 (left). Another group of WT mice were given OVA–Alexa Fluor 647 i.n., and OVA-loaded lung tissue MØs and MLN DCs were isolated. These secondary APCs were then co-cultured with purified OT-II T cells that had been activated with MLN DCs at a 1:10 APC/T ratio. Induction of Foxp3 was assessed after 4 d of secondary culture (right). Data are representative of three independent experiments.

Article Snippet: The following antibodies were purchased from BD: anti–CD4-allophycocyanin, anti–CD4-PerCp, anti–CD4-FITC, anti–CD11c-FITC or -allophycocyanin, anti–CD11b-FITC or -allophycocyanin, anti-CD24–Alexa Fluor 647, anti–CD45-PerCp, anti–CD45.2-FITC or -allophycocyanin, anti–CD45.1-FITC or -allophycocyanin, anti–CD62L-FITC, anti-CD68–Alexa Fluor 647, anti-CD205–Alexa Fluor 647, anti–I-A/I-E–PE, anti–Vα2-PE, anti–Vβ5-FITC, and anti–Siglec F–PE.

Techniques: Cell Culture, Activation Assay, Isolation, Purification