Percp cy5.5 anti cd14

For FCGRs detection, primary B cells, monocytes, and macrophages were incubated with fluorochrome-labeled antibodies specific for humans before fixation: FITC mouse anti-human CD32 (552883; BD Pharmingen, San Diego, CA, USA), APC mouse anti-human CD64 (561189; BD Pharmingen, San Diego, CA, USA ), CD16 Rabbit PAb (16559-1-AP; Proteintech,--). FITC-anti-Rabbit IgG (H+L) (SA00003-2; Proteintech, Chicago, IL, USA) was used as the secondary antibody for CD16.
For SARS-CoV-2 infected primary immune cells, surface staining was conducted before fixation with AF700-anti-CD45 (368514; BioLegend, San Diege, CA, USA), BV650-anti-CD11b (101239; BioLegend, San Diege, CA, USA), PE-anti-CD68 (333808; Biolegend, San Diege, CA, USA), and Percp Cy5.5-anti-CD14 (367110; Biolegend, San Diege, CA, USA). Antibody stained cells were fixed overnight with 4% PFA at 4 °C and taken out of the BSL3 lab for downstream analysis. Cells were stained further with in house made SARS-CoV NP pAb (1:500) at 4 °C for 30 min after permeabilization. Then, cells were stained with FITC-anti-Rabbit IgG (H + L) (SA00003-2; Proteintech, Chicago, IL, USA) at room temperature for 30 min.

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).