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3 HLA independent killing of cervical tumors by UCB-NK cells | 85 flow cytometer LSR Fortessa (BD Biosciences). Phenotypic analyses were obtained from at least two independent experiments performed on each cell line. Data were analyzed using Kaluza software (Beckman coulter) and calculated as specific (geometric) mean fluorescence intensity (MFI) (MFI; geometric mean fluorescence of marker - geometric mean fluorescence of isotype). RAS typing RAS status was obtained from Rational molecular Assessments and Innovative Drugs selection (RAIDs) project data (http://www.raids-fp7.eu/project-overview.html ) and www. lgcstandards-atcc.org for cell lines HeLa, SiHa, CaSki, C33A, CSCC7, CC10A and CC10B. In addition, full RAS typing (i.e., BRAF exon 15, KRAS exon 2-3-4 and NRAS exon 2-3-4) was performed for cell lines CC8, CC11A and CC11B at the molecular pathology lab of the Department of Pathology of the VUUniversitymedical center (Amsterdam, TheNetherlands) using high resolution melting assay followed by Sanger sequencing of using high resolution melting-PCR products with an aberrant melt curve, essentially as described previously 36,37 . PBMC isolation & NK cell isolation Whole blood samples fromfour healthy volunteerswere collected. PBMCwere isolated using Lymphoprep™ (STEMCELL Technologies, The Netherlands) density gradient centrifugation. CD56 + NK cells were isolated from PBMC using a MACS ® Human NK cell isolation kit (Miltenyi Biotech, Bergisch Gladbach, Germany) according to the manufacturer’s instructions. The cell number and purity of the isolated PBNK was analyzed by flow cytometry. Isolated NK cells were activated overnight with 1000 U/ml IL-2 (Proleukin ® ; Chiron, München, Germany) and 10 ng/ml IL-15 (CellGenix) before use in cytotoxicity assays. NK cell purity and viability were checked by flow cytometry using the following antibodies: 7-Aminoactinomycin D (7AAD; Sigma Aldrich), CD3 (labeled with VioBlue), CD56 (labeled with APC-Vio770), and CD16 (labeled with APC) (all from Miltenyi Biotech). Purity of NK cells obtained from NK donors was 87± 6%. For cytotoxicity assays, only PBNK with CD16 expression rates exceeding 80% were used. UCB-NK isolation and cultures Allogeneic NK cells were generated from cryopreserved umbilical cord blood hematopoietic stem cells as previously described 38 . CD34 +  UCB cells (3×10 5  per ml) were plated into 12- well tissue culture plates (Corning Incorporated, Corning, NY) in Glycostem Basal Growth Medium (GBGM ® ) (Clear Cell Technologies, Beernem, Belgium) supplemented with 10% human serum (Sanquin Bloodbank, The Netherlands), 25 ng/mL of SCF, Flt-3L, TPO, and IL-7 (CellGenix, Germany). In the expansion phase II, from day 9 to 14, TPO was replaced