Nk cell microbead cocktail

Adult PB was obtained as buffy coat products from healthy adult donors from the
New York Blood Center. Umbilical CB was obtained by venipuncture from umbilical
cord veins immediately after infant delivery at Morgan Stanley Children’s
Hospital of New York-Presbyterian Hospital. This protocol was approved by the
Columbia University Human Subjects Institutional Review Board (approval number
0944), and written informed consent was obtained from parents/legal guardians,
and patients if over 18 yrs of age, in accordance with the Declaration of
Helsinki. PB and CB mononuclear cells (MNC) (n = 5 of each)
were isolated following Ficoll-Paque (Amersham Biosciences, Piscataway, NJ, USA)
density gradient separation. Pure NK cells (CD3^-–/56⁺)
were isolated by indirect magnetic labeling system with a mixture of
biotin-conjugated Abs and the NK Cell MicroBead Cocktail using a standard kit
(Miltenyi Biotec, Auburn, CA, USA). After magnetic separation of NK
(CD3^–/56⁺) cells, cells were further isolated into
CD3^–56^dim and NK CD3^–56^brightsubsets using a cell sorter (FACS Aria flow cytometer/cell sorter, BD
Biosciences, San Jose, CA, USA). After sorting, four distinct NK cell
populations were obtained: PB CD56^dim/CD16^bright, PB
CD56^bright/CD16^bright, CB
CD56^dim/CD16^bright, and CB
CD56^bright/CD16^bright, with 99.9% purity of each
subset.

Human peripheral blood mononuclear cells (PBMCs) were isolated from human buffy coats from the New York Blood Center by density gradient centrifugation using Ficoll-Paque PLUS (GE Healthcare). Primary NK cells were extracted from PBMCs by negative immunomagnetic isolation using a NK cell isolation kit (Miltenyi Biotec) according to the manufacturer’s protocol. Briefly, cells were incubated with NK Cell Biotin-Antibody Cocktail and NK Cell MicroBead Cocktail to label non-NK cells, followed by loading onto a LS Separation column (Miltenyi Biotec). The cell suspension was then placed in the magnetic field of a MACS Separator to separate labeled from non-labeled cells. The isolated human NK cells were subsequently cultured in Roswell Park Memorial Institute (RPMI) 1640 media supplemented with 10% fetal bovine serum, 1x penicillin/streptomycin and 100 U/mL IL-2 at 37°C in 5% CO₂. The purity of the NK cell population was validated by flow cytometry using PE-Vio 770 anti-human CD56 (1:50; REA196 clone; Miltenyi Biotec) and FITC anti-human CD3 (1:50; REA613 clone; Miltenyi Biotec) antibodies.