Pe cy7 anti cd123 clone 7g3

Single cell suspensions from surgical excess colon tissue or HIV-uninfected control PBMCs were stained for viability with a LIVE/DEAD fixable violet dead cell stain kit (Life Technologies). For phenotypic characterization of epithelial and tissue cells, cell surface antigens were stained as described above with the following mouse anti-human monoclonal antibodies: APC anti-CD326 (clone EBA-1; BD Bioscience), v500 anti-CD45 (clone HI30; BD Bioscience). For phenotypic characterization of monocytes and DCs from PBMCs, cell surface antigens were stained for 15 min at room temperature with the following mouse anti-human monoclonal antibodies: FITC anti-CD3 (clone UCHT1; Biolegend), CD19 (clone HIB19; Biolegend), CD66b (clone G10F5; BD Bioscience), v500 anti-CD45 (clone HI30; BD Bioscience), APC-H7 anti-CD16 (clone 3G8; BD Bioscience), AF700 anti-HLA-DR (clone L243; BD Bioscience), APC anti-CD11c (clone B-ly6; BD Bioscience), PE-Cy7 anti-CD123 (clone 7G3; BD Bioscience), PerCP-Cy5.5 anti-CD14 (clone M5E2; Biolegend). Stained cells were resuspended in PBS with 5 mM EDTA and sorted using a FACS Aria II (BD Biosciences).

Cryopreserved PBMCs from acute (n = 8) and HIV-uninfected control subjects (n = 9) were thawed and 0.5x10⁶ cells were stained for viability with a LIVE/DEAD fixable blue or green dead cell stain kit (Life Technologies). For phenotypic characterization of monocytes and DCs, cell surface antigens were stained for 15 min at room temperature with the following mouse anti-human monoclonal antibodies: FITC anti-CD3 (clone UCHT1; Biolegend), CD19 (clone HIB19; Biolegend), CD56 (clone HCD56; Biolegend), CD66b (clone G10F5; BD Bioscience), BV605 anti-CD4 (clone SK3; BD Bioscience), v500 anti-CD45 (clone HI30; BD Bioscience), APC-H7 anti-CD16 (clone 3G8; BD Bioscience), PE-Cy5 anti-CXCR4 (clone 12G5; BD Bioscience), AF700 anti-HLA-DR (clone L243; BD Bioscience), APC anti-CD11c (clone B-ly6; BD Bioscience), PE-Cy7 anti-CD123 (clone 7G3; BD Bioscience), BV421 anti-CCR5 (clone 2D7; BD Bioscience), PerCP-Cy5.5 anti-CD14 (clone M5E2; Biolegend). The cells were fixed with 2% paraformaldehyde before running on a LSR Fortessa flow cytometer (BD Biosciences) within 4 h. Flow data were analyzed with FlowJo (TreeStar).