Hct116

The human MCF-7 breast adenocarcinoma cell line was purchased from the European Collection of Cell Cultures (ECACC; Salisbury, UK). Human HL-60 promyelocytic leukemia cells, human HCT116 colorectal carcinoma cells, human A549 lung adenocarcinoma cells, the H9c2 cell line, derived from embryonic rat heart tissue, and 3T3 mouse embryo fibroblasts were obtained from the American Type Culture Collection (ATCC; Manassas, VA, USA). The MCF-7, HCT116, A549, 3T3 and H9c2 cell-types were cultured in Dulbecco’s modified Eagle’s medium (DMEM; Lonza, Basel, Switzerland). In the case of MCF-7 cells, DMEM was used without phenol red. DMEM was supplemented with 10% (v/v) heat-inactivated fetal bovine serum (FBS; Lonza), 1% penicillin/streptomycin solution (Lonza) and 10 mM HEPES buffer (pH 7.0–7.6; Sigma-Aldrich). The HL-60 cell line was maintained in RPMI medium (Sigma-Aldrich) supplemented with 10% heat-inactivated FBS and 1% penicillin/streptomycin solution. All cell lines were cultured in 75 cm² tissue culture flasks (TPP, Trasadingen, Switzerland) at 37°C in a humidified atmosphere of 5% CO₂. Sub-confluent adherent cells, or the suspension of HL-60 cells, were sub-cultured every 3–4 days.

For assessment of NK cell function, donor control PBMCs or CBMCs were plated in RPMI (Lonza, Slough, UK) containing 10% heat-inactivated fetal calf serum (FCS) supplemented with 1% penicillin and streptomycin (complete media) and containing human IL-2. Test cultures using 50% CBP diluted with complete media or complete media only contained 200 IU IL-2/well in 96-well plates. All cultures were incubated at 37°C with 5% CO₂ for 48 h before PMA and ionomycin stimulation. Duplicate cultures were carried out for each experiment without PMA and ionomycin stimulation to assess levels of NKG2D and baseline CD107a. NKG2D expression on the relevant cells was determined using the unstimulated cultures and CD107a background levels in unstimulated cultures were subtracted from the values obtained for equivalent stimulated cells.
Experiments were carried out using four different, healthy PBMC donors, each tested with CBP samples from the 181 UCB units and data points represent donor means. For experiments using CBMCs, four CB units were used with seven different CBP samples. HCT116 (human colon carcinoma) used for luciferase assays were obtained from the European Collection of Cell Cultures (ECACC, Salisbury, UK). HCT116 cells were cultured in McCoy’s 5A medium (Lonza, Slough, UK) supplemented with 2 mM l-glutamine and 10% FCS at 37°C with 5% CO₂.