The human OC cell lines, WSU-HN4 (donated by the University of Maryland) and SCC-9 (purchased from ATCC, CRL-1629) were cultured in Dulbecco's modified Eagle's medium (DMEM; Gibco; Thermo Fisher Scientific, Inc.) and DMEM/F-12 (1:1) (basal medium, Gibco; Thermo Fisher Scientific, Inc.), respectively. The human NK cell line NK92MI (purchased from ATCC, CRL-2408) was cultured in RPMI-1640 (basal medium, Gibco; Thermo Fisher Scientific, Inc.). All media were supplemented with 10% fetal bovine serum (FBS; Bovogen Biologicals Pty Ltd.) and 1% penicillin-streptomycin (Sigma-Aldrich; Merck KGaA), and all cells were incubated in a humidified incubator with 5% CO2 at 37°C. In all OCEX stimulation experiments, OCEXs were co-cultured with NK92MI cells for 1, 3 and 7 days. In all transforming growth factor (TGF)-β1 stimulation experiments, recombinant human TGF-β1 (PeproTech, Inc.) was used at a concentration of 10 ng/ml for 1, 3 and 7 days, as previously described (32 (link)-34 (link)).
The SCC-9 cells were seeded in a 96-well plate at a density of 1,000 cells per well. Approximately 24 h later, the tumor cells were co-cultured with the NK92MI cells in medium without FBS and penicillin-streptomycin for 4 h, after which the culture supernatant was removed. The wells were washed gently with PBS and observed under an inverted-phase microscope (Zeiss AG).
The SCC-9 cells were seeded in a 96-well plate at a density of 1,000 cells per well. Approximately 24 h later, the tumor cells were co-cultured with the NK92MI cells in medium without FBS and penicillin-streptomycin for 4 h, after which the culture supernatant was removed. The wells were washed gently with PBS and observed under an inverted-phase microscope (Zeiss AG).