Isolation of Immune Cells for COVID-19 Research

Enriched leukocytes were obtained from the New York Blood Center (Long Island City, NY) after informed consent of donors who were deemed healthy by the New York Blood Center’s criteria and used under a protocol approved by the Institutional Review Board of the Hospital for Special Surgery and the Institutional Biosafety Committee of Weill Cornell Medicine (2014–221). PBMCs were prepared using Ficoll-Paque density gradient (GE Healthcare) as previously described (78 ). pDCs and monocytes were isolated from PBMCs by positive selection using BDCA4-conjugated (78 ) and CD14-conjugated (79 (link)) microbeads (Miltenyi Biotec), respectively. pDC-depleted PBMCs were prepared by removing BDCA4-positve cells from PBMCs using microbeads (Miltenyi Biotec). Macrophages were differentiated from monocytes by culturing in complete RPMI 1640 medium with macrophage colony-stimulating factor (M-CSF; 20 ng/ml) for 1 to 5 days. Vero E6 cells [African green monkey (Chlorocebus aethiops) kidney] were obtained from the American Type Culture Collection (ATCC). A549 cells (human adenocarcinomic alveolar basal epithelial) were obtained from ATCC. A549-ACE2 cells were obtained from B.R.t. (Mount Sinai).