Figure 1. Characterization of resident... | Rockefeller University Press

$Figure 1. Characterization of resident dermal APC. (A) Strategy used in the characterization of resident CD45+HLA-DR+ dermal cells showing successive gating on DAPI-live cells, CD45+ cells, and HLA-DR+ cells. Progressive refinement of SSCloAFlo and SSChiAFhi fractions is depicted at each step by the corresponding panel below. CD45+ cells include macrophages, DC, mast cells, and lymphocytes. Mast cells (high SSC) are HLA-DR−, as are the majority of lymphocytes (low SSC), and are easily excluded by the final HLA-DR gate. AF was recorded in the FL1 channel (488-nm laser and 530/30 band-pass filter). Similar results were found in >50 independent preparations of skin. (B) Further analysis of SSCloAFlo and SSChiAFhi fractions of CD45+HLA-DR+ cells by expression of CD14+ and CD1a+. Blue indicates the position of isotype controls and red the expression of CD14/CD1a by each fraction. Three principal populations were sorted and show distinct morphological appearances by Giemsa staining (right). Bar, 20 µm. A representative example of three experiments is shown. (C) Surface phenotype of resident CD45+HLA-DR+ dermal cells. Gates were placed as described in A and B using CD45, HLA-DR, SSC, AF CD1a, and CD14 to define macrophages (mac), CD14+ DC, and CD1a+ DC. CD14 expression on DC cells was analyzed by gating on CD1a+ and CD1a− cells. All other markers for CD1a+ DC and CD14+ DC were determined by gating on CD14− and CD14+ populations, respectively. Staining was performed at least six times on different skin preparations with similar results. (D) Identification of large melanosome-laden macrophages by CD45, HLA-DR, and FXIIIa. Freshly isolated APC prepared on cytospin were simultaneously stained with antibodies to CD45 (green), HLA-DR (red), and FXIIIa (blue). After immunofluoresence imaging the coverslip was removed and the slide stained with Giemsa. FXIIIa staining is restricted to macrophages that are a subset of the HLA-DR+ APC in the preparation (red). A number of small agranular HLA-DR+ DC with reniform nuclei are FXIIIa negative. All HLA-DR+ cells and a number of small lymphocytes in the field are highlighted by CD45 staining. Bar, 20 µm. This finding was reproduced three times.$

Figure 1.

Characterization of resident dermal APC. (A) Strategy used in the characterization of resident CD45⁺HLA-DR⁺ dermal cells showing successive gating on DAPI-live cells, CD45⁺ cells, and HLA-DR⁺ cells. Progressive refinement of SSC^loAF^lo and SSC^hiAF^hi fractions is depicted at each step by the corresponding panel below. CD45⁺ cells include macrophages, DC, mast cells, and lymphocytes. Mast cells (high SSC) are HLA-DR⁻, as are the majority of lymphocytes (low SSC), and are easily excluded by the final HLA-DR gate. AF was recorded in the FL1 channel (488-nm laser and 530/30 band-pass filter). Similar results were found in >50 independent preparations of skin. (B) Further analysis of SSC^loAF^lo and SSC^hiAF^hi fractions of CD45⁺HLA-DR⁺ cells by expression of CD14⁺ and CD1a⁺. Blue indicates the position of isotype controls and red the expression of CD14/CD1a by each fraction. Three principal populations were sorted and show distinct morphological appearances by Giemsa staining (right). Bar, 20 µm. A representative example of three experiments is shown. (C) Surface phenotype of resident CD45⁺HLA-DR⁺ dermal cells. Gates were placed as described in A and B using CD45, HLA-DR, SSC, AF CD1a, and CD14 to define macrophages (mac), CD14⁺ DC, and CD1a⁺ DC. CD14 expression on DC cells was analyzed by gating on CD1a⁺ and CD1a⁻ cells. All other markers for CD1a⁺ DC and CD14⁺ DC were determined by gating on CD14⁻ and CD14⁺ populations, respectively. Staining was performed at least six times on different skin preparations with similar results. (D) Identification of large melanosome-laden macrophages by CD45, HLA-DR, and FXIIIa. Freshly isolated APC prepared on cytospin were simultaneously stained with antibodies to CD45 (green), HLA-DR (red), and FXIIIa (blue). After immunofluoresence imaging the coverslip was removed and the slide stained with Giemsa. FXIIIa staining is restricted to macrophages that are a subset of the HLA-DR+ APC in the preparation (red). A number of small agranular HLA-DR+ DC with reniform nuclei are FXIIIa negative. All HLA-DR⁺ cells and a number of small lymphocytes in the field are highlighted by CD45 staining. Bar, 20 µm. This finding was reproduced three times.

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