Anti human cd32 fc gamma rii blocker

NK cells were isolated from Buffy Coats obtained from healthy donors using the EasySep Human FITC Positive Selection Kit II (Stemcell Technologies) to deplete CD3⁺, CD14⁺, and CD19⁺ cells in the presence of anti-human CD32 (Fc gamma RII) Blocker (Stemcell Technologies). Subsequently, the remaining cells were stained with CD3 FITC (Beckton Dickinson), CD56 PE-Cy7, CD45 Krome Orange (both Beckman Coulter), and LIVE/DEAD Fixable Near-IR Dead Cell fluorescent dye (Invitrogen). Lineage negative and CD56⁺ NK cells were sorted on the FACSaria IIu (Beckton Dickinson), and the purity of NK cells (>95%) was checked during the procedure.

To perform cell sorts, blood DCs were enriched with the EasySep Pan-DC Pre-Enrichment Kit (Stemcell Technologies) as described in the manufacturer’s manual. Briefly, PBMCs were diluted in PBS+2% human sera+1 mM EDTA and stained with anti-human CD32 (Fc gamma RII) Blocker (Stemcell Technologies) and the EasySep Pan-DC Pre-Enrichment Cocktail for 45 min at room temperature (RT). Then, magnetic Dextran Rapidspheres were added, and cells were incubated for 10 min at RT followed by incubation for 5 min inside “The Big Easy” EasySep Magnet (Stemcell Technologies). The unlabeled DCs were poured off into a new tube and stained for cell sorts.