Anti cd56 microbeads

uNK cells were purified as previously described (2 (link)). Briefly, decidual tissues were thoroughly washed with Ca²⁺- and Mg²⁺-free Hank’s balanced salt solution (HBSS) containing 100 U/ml penicillin and 100 g/ml streptomycin (Sigma-Aldrich, St. Louis, MO, USA), cut into fragments of 1–2 mm³ using two scalpels and digested for 1 h at 37°C with gentle agitation in HBSS with 0.1% (w/v) collagenase I (Gibco-BRL, Carlsbad, CA, USA). Cell suspensions were layered over Ficoll-Hypaque medium (General Electric, Fairfield, CT, USA) and centrifuged at 800 × g for 25 min. Cells at the interface were washed twice in RPMI-1640 media with 10% fetal calf serum (FCS) and antibiotics. Following incubation for 20 min at 4°C with anti-CD56 micro beads (Miltenyi Biotec GmbH, Bergisch Gladbach, Germany), cells were washed in washing buffer [phosphate-buffered saline (PBS), EDTA 2 mM and 0.5% bovine serum albumin(w/v)] and loaded onto a manual cell separation (MS) column in a MiniMACS magnet (MiniMACS^TM Separator System; Miltenyi Biotec GmbH). The MS column was flushed three times and CD56⁺ cells were flushed according to the manufacturer’s instructions. The purity of the uNK cells was >90% CD56⁺CD3⁻ according to flow cytometric analysis. The uNK cells were cultured in RPMI-1640 media with 1% FCS and 10 ng/ml IL-15 (R&D Systems Inc., Minneapolis, MN, USA).