Negative selection nk cell isolation kit

Peripheral blood mononuclear cells (PBMCs) were purified by ficoll density centrifugation using LSM1077 (Sigma-Aldrich). NK cells were isolated using a negative selection NK cell isolation kit (Miltenyi Biotec, Bergisch Gladbach, Germany). Isolated NK cells were cultured in IMDM medium (Gibco^TM) with 10% FCS, 1% P/S, and 10 U/mL IL-2 and rested at 37 °C overnight. NK cells were pretreated with 100 µg/mL EVs or soluble protein for 24 h before killing assays were performed. For killing assays, K562 target cells were stained with 5 µM CellTracker^TM Violet BMQC (Life Technologies) fluorescent dye in serum-free medium at 37 °C for 45 min. Then, FCS was added to the cells at a final concentration of 20%. Cells were resuspended in fresh medium and seeded accordingly in IMDM medium with 10% FCS and 1% P/S in U-shaped, 96-well plates. NK cells were added to the target cells in ratios ranging from 1.25:1 to 10:1. The cells were cocultured for 3 h before they were harvested and centrifuged at 300× g for 5 min. The supernatant was discarded, and the cells were resuspended in 200 µL PBS before they were stained with 100 ng/200 µL 7-AAD (BioLegend). Cell death was measured by flow cytometry using a FACS Canto II cytometer (BD Bioscience) and analyzed by FACS Diva software. The killing of untreated NK cells was subtracted from the treated samples and induced killing was displayed.

Cell lines: The human pancreatic cancer cell lines Panc-1 and PaTu8988t cells were purchased from ATCC. The cells were cultured in DMEM with 10% FCS and 1% P/S at 37 °C and 5% CO₂.
NK cells: Leukocytes from leukoreduction system chambers of healthy donors were provided by the blood bank of the University Hospital of Giessen and Marburg. Peripheral blood mononuclear cells (PBMCs) were purified by ficoll density centrifugation followed by a centrifugation step at 10 g for 5 min to deplete the sample of monocytes. NK cells were then isolated using a negative selection NK cell isolation kit (Miltenyi Biotec, Bergisch Gladbach, Germany). Isolated NK cells were cultured in IMDM medium with 10% FCS, 1% P/S, 100 ng/mL IL-15 and 200 U/mL IL-2 and rested at 37 °C overnight before killing assays were performed.

Peripheral blood mononuclear cells (PBMCs) were purified by ficoll density centrifugation using LSM1077 (Sigma-Aldrich). NK cells were isolated using a negative selection NK cell isolation kit (Miltenyi Biotec, Bergisch Gladbach, Germany). Isolated NK cells were cultured in IMDM medium (Gibco^TM) with 10% FCS, 1% P/S, and 10 U/mL IL-2 and rested at 37 °C overnight. For killing assays, HL60 cells were stained with 5 µM CellTracker^TM Violet BMQC (Life Technologies) fluorescent dye in serum-free medium at 37 °C for 45 min. Then, FCS was added to the cells at a final concentration of 20%. Cells were resuspended in fresh medium and seeded accordingly in IMDM medium with 10% FCS and 1% P/S in U-shaped 96-well plates. NK cells were added to the target cells in ratios ranging from 1.25:1 to 10:1. The cells were co-cultured for 3 h before they were harvested and centrifuged at 300 × g for 5 min. The supernatant was discarded, and the cells were resuspended in 200 µL PBS before they were stained with 1 µl 50 µg/ml PI per sample. Cell death was measured by flow cytometry using a FACS Canto II cytometer (BD Bioscience) and analyzed by FACS Diva software.