Ls cell separation column

PBMCs were isolated from healthy donors via density gradient centrifugation using Ficoll-Paque Plus medium (GE Healthcare) and washed with Ca/Mg-free PBS. The PBMC fraction was then treated with red blood cell lysis buffer (BioLegend) to remove red blood cells and washed with Ca/Mg-free PBS containing 2% FBS. Isolated PBMCs were then subjected to the purification of monocytes by using the Pan-monocyte Isolation Kit (Miltenyi), according to the manufacturer’s instructions. Briefly, PBMCs were blocked with 10 μl of Fc receptor–blocking reagent, stained with 10 μl of biotin-antibody cocktail for 10 min at 4°C, and subsequently stained with 20 μl of antibiotin microbeads (Miltenyi) for 15 min at 4°C. Then, PBMC suspension was infused through an LS cell separation column (Miltenyi) placed in the MACS Separator (Miltenyi), and the negative fraction containing enriched and unlabeled monocytes was collected and counted. The purity of the isolated monocyte populations was determined by flow cytometry using the markers CD45, CD14, and CD16 and was found to be >95% pure. The surface expression of CCR2 on purified monocytes was examined by flow cytometry analysis. Monocytes were infected with SFTSV at an MOI of 10 for the virus binding assay as mentioned above.