Dmem f12 medium

The human colorectal carcinoma (CRC) cell line SW620 was obtained from the cell bank of the IRCCS Ospedale Policlinico San Martino, Genoa (kind gift of Dr. Alessandro Poggi MD, Head of the Molecular Oncology and Angiogenesis Unit, IRCCS Ospedale Policlinico San Martino, Genoa, Italy). SW620 cell line was cultured in RPMI 1640 (Corning, New York, NY, USA) medium supplemented with 10% fetal bovine serum (FBS, Gibco™, Thermo Fisher Scientific Italy, Monza, Italy), penicillin/streptomycin and l-glutamine (Corning, New York, NY, USA) in a humidified incubator at 37 °C with 5% CO₂.
For the generation of tumor spheroids, SW620 adherent cells were detached with Trypsin/EDTA (Corning, New York, NY, USA) and counted using a standard hemocytometer.
Then, SW620 cell suspension was seeded at the concentration of 1.8 × 10⁴ cells per well in flat-bottom 96-well plate (Corning^®Costar^®, New York, NY, USA) pre-treated with Poly-HEMA 3% in EtOH 96% (P3932, Sigma Aldrich, Merck, Milano, Italy) to prevent cell adhesion, in serum-free DMEM-F12 medium (Euroclone, Milan, Italy), supplemented with epithelial growth factor (EGF) (Peprotech Europe, London, UK) at 10 ng/mL final concentration (≥1 × 10⁶ U/mg). Experiments were performed at proper spheroids compaction, after approximately 1 week of culture.

High-grade serous ovarian cancer cell lines PEA1, PEA2, PEO14, and PEO23 were purchased from Sigma-Aldrich (St. Louis, MO, USA) and were grown in RPMI-1640 medium supplemented with 10% fetal bovine serum, 2 mM glutamine, 2 mM sodium pyruvate, and 1% penicillin/streptomycin (Euroclone S.P.S., Pero, Italy). High-grade serous ovarian cancer cell lines KURAMOCHI and OVSAHO were obtained from the Japanese Collection of Research Bioresources Cell Bank (JCRB). The human ovarian cancer cell lines SKOV3, HeyA8 (High-grade serous ovarian cancer cell line), and TOV21G were provided by the CEINGE Cell Culture Facility (Naples, Italy). All these cell lines were maintained in RPMI-1640 medium supplemented with 10% fetal bovine serum and 1% penicillin/streptomycin (Euroclone S.P.S., Pero, Italy). The immortalized fallopian tube secretory epithelial cell line FT194 was provided by Dr. R. Drapkin (Boston, MA, USA). This cell line was grown in DMEM-F12 medium (Euroclone S.P.S., Pero, Italy) supplemented with 2% Ultroser G serum (PALL, Cergy-Saint-Christophe, France) and 1% penicillin/streptomycin. Primary human fallopian tube secretory epithelial cells were provided by Dr. U. Cavallaro (Milan, Italy) and was grown in appropriate medium [38 (link)].

HeLa cell line (ATCC CCL-2) was grown in DMEM F12 medium (Euroclone) supplemented with L-glutamine in presence of 1% penicillin-streptamycine and 10% of FCS at 37°C with the 5% of CO2. HeLa cell lines were infected with HHV-6A and HSV-1 to evaluate NK cell activation. We used HeLa cells as a good in vitro model of epithelial cells to perform and obtain HSV-1 and HHV-6A in vitro infection.
We used HHV-6A (strain U1102) cell free virus inocula [30 (link)] and infected with 10 genome equivalents per 1 cell. We used HSV-1 strain F at a multiplicity of infection of 0.1 PFU (plaque forming unit)/cell for 48 hrs [31 (link)]. We used HSV-1 and HHV-6A UV-inactivated viral preparations as controls.
Infected cells were then collected to perform co-culture experiment with NK cells.