Easysep human monocytes enrichment kit without cd16 depletion

Monocytes were isolated from PBMCs (purchased from Astarte Biologics, Inc.) using EasySep human monocytes enrichment kit without CD16 depletion (StemCell Technologies, Inc.) as per the manufacturer's instructions. The viability of cells after isolation was 90% and the purity was ≥95% according to flow cytometry analysis staining with human CD14 antibody. One million of monocytes were nucleofected with 10 μg of pLVX-PD-1H plasmid (or pLVX vector plasmid as negative control), or siPD-1H (and siScramble as negative control). The above experiments were duplicated. Lonza 4D nucleofection system was used for the nucleofection according to parameters recommended by the manufacturer. The nucleofected monocytes was cultured in PBMC supplemented with 5% human serum (heat-inactivated) for 24 h. Then, the cells were harvested and sorted by FACS using the endogenous GFP expression by the pLVX vector. The GFP-positive cells were then used for RNA extraction. Monocytes were isolated from three different donors, and the experiments were repeated twice each time with the same three donors.