Penicillin streptomycin solution

Blood samples were collected in 3-mL K3 EDTA vacutainer tubes (Cat. No. 368857, Becton Dickinson, Heidelberg, Germany). Peripheral blood mononuclear cells (PBMCs) were isolated via density gradient centrifugation in a Biocoll Separating Solution (Cat. No. L6115, Biochrom GmbH, Berlin, Germany) within 2 h of sample collection. PBMcs were plated in RPMI-1640 (Life Technologies, Waltham, MA, USA) supplemented with a 10% serum and 1% penicillin-streptomycin solution (Aurogene, Rome, Italy) for 12 h in a 37C incubator in a humidified atmosphere of 5% CO₂ and 95% air. After adhesion of the monocytes and dentritic cells, PBLs (peripheral blood lymphocytes) were derived and subsequently used for the experiments.

The human HCC HUH7, human embryonic kidney HEK293T and human prostate cancer PC3 cell lines were cultured in DMEM (Life Technologies, Inc., Grand Island, NY), the primary human dermal fibroblast HDFa, human ovarian carcinoma OVCAR3, and human glioblastoma ADF cell lines were cultured in RPMI-1640 (Life Technologies). Culture media were supplemented with 10% fetal bovine serum and a 1% penicillin-streptomycin solution (Aurogene, Rome, Italy). The cells were cultured at 37 °C in a humidified atmosphere of 5% CO₂ and 95% air. Cell lines were obtained from the ATCC (Georgetown University in Washington, DC), excluding the ADF cell line, which was kindly provided by Dr. Marco Paggi (Regina Elena Cancer Institute, Rome, Italy).