Cd3 fitc antibodies

Human PBMCs and NK cells were freshly isolated from leukopaks (American Red Cross, Columbus, OH) as described previously (41 (link)). PBMCs were isolated by Ficoll-Paque Plus (GE Healthcare Bio-Sciences, Pittsburgh, PA) density gradient centrifugation. NK cells (CD56⁺CD3⁻) were enriched with RosetteSep NK cell enrichment mixture (StemCell Technologies, Vancouver, Canada). The purity of enriched NK cells was ≥80 % (data not shown), assessed by flow cytometric analysis after staining with CD56-APC and CD3-FITC antibodies (BD Biosciences, San Jose, CA). These enriched NK cells were further purified with CD56 magnetic beads and LS columns (Miltenyi Biotec, Auburn, CA). The purity of magnetic bead-purified NK cells was ≥ 99.5% (data not shown), as determined by the aforementioned flow cytometric analysis. CD56^bright and CD56^dim NK cell subsets were sorted by a FACS Aria II cell sorter (BD Biosciences) based on CD56 cell-surface density after staining with CD56-APC and CD3-FITC antibodies. The purity of CD56^bright and CD56^dim subsets was ≥ 99.0% (data not shown). All human work is approved by The Ohio State University Institutional Review Board.