Anti cd19 dynabeads

Buffy coats were obtained from healthy human donors from the blood bank at Oslo University Hospital according to the Declaration of Helsinki. The study was approved by the South-Eastern Norway Regional Ethical Committee (REK2012-1452). Primary NK cells were enriched through density gradient separation using RosetteSep NK cell Enrichment Cocktail according to the manufacturer’s protocol (StemCell Technologies), followed by B-cell depletion using anti-CD19 Dynabeads (ThermoFisher Scientific). The resulting NK cell population was > 90% CD56⁺ and CD3⁻. The following tumor cell lines obtained from ATCC were used: NK-92 (NK cell line), HCT116 (colorectal carcinoma), HCT-15 (Dukes type C colorectal adenocarcinoma), DU145 (prostate carcinoma), PC3 (prostate adenocarcinoma), SK-BR-3 (breast adenocarcinoma), T-4D7 (mammary gland ductal carcinoma), OVCAR-3 (ovarian adenocarcinoma), WM9 (metastatic melanoma), and U87 (glioblastoma). The NK cell line KHYG-1 was obtained from the German Collection of Microorganisms and Cell Cultures (DSMZ). All cell lines were cultured in complete RPMI-1640 medium (cRPMI), supplemented with 10% FBS, 1% penicillin/streptomycin, 1% sodium pyruvate, and 50 mM 2-mercaptoethanol (NK-92 in cRPMI with 20% FBS). NK-92 and KHYG-1 cell cultures were supplemented with 500 IU/ml human recombinant IL-2.

Buffy coats from healthy human donors were obtained from the blood bank at Oslo University Hospital according to the Declaration of Helsinki. The study was approved by the South-Eastern Norway Regional Ethical Committee (REK2012-1452). Primary NK cells were enriched using RosetteSep NK cell Enrichment Cocktail according to manufacturer’s protocol (Stemcell Technologies), and B cells were depleted by using anti-CD19 Dynabeads (ThermoFisher Scientific). Final NK cell purity was >90% CD56⁺ and CD3^-. The NK-92 cell line was obtained from ATCC (CRL-2407), and was cultured in complete RPMI medium (RPMI-1640 containing 20% FBS, 1% penicillin/streptomycin, 1% sodium pyruvate, and 50 mM 2-mercaptoethanol) supplemented with 500 IU/ml human recombinant IL-2 (R&D Systems). The HCT-116 colorectal carcinoma cell line was cultured in cPRMI with 10% FBS, and split every 2-3 days.

Blood samples were collected from donors and peripheral blood mononuclear cells (PBMCs) were isolated by Ficoll-Hypaque centrifugation. Lymphoblastoid cell lines (LCLs) were generated by culturing PBMCs in standard LCL culture media (RPMI 1640 medium with 100 IU/ml penicillin, 100 mg/ml streptomycin, L-glutamine and 10% foetal calf serum) supplemented with 0.1ug/ml cyclosporin A (CSA; Sandimmune; Novartis Pharmaceuticals) with the addition of B95.8 strain EBV or a recombinant EBV lacking the BZLF1 gene (to generate B95.8 or BZLF1-K/O LCLs respectively). B-cells were isolated from PBMC immediately before use in experiments using anti-CD19 Dynabeads, and CD19 Detach-a-beads (Invitrogen) following the manufacturer’s instructions. B-cell purity was assessed by flow cytometry and purified B-cell preparations typically contained at least 95% B-cells.