Penicillin pen

Human dermal fibroblasts were obtained from the American Type Culture Collection (Manassas, VA, USA); maintained in Dulbecco’s Modified Eagle Medium (DMEM) (PANTM BIOTECH, Aiden Bach, Germany); supplemented with 10% Fetal Bovine Serum (FBS) (Gibco, Waltham, MA, USA), 50 U/mL of Penicillin (Pen) (Gibco, Waltham, MA, USA), and 50 μg/mL of Streptomycin (Strep) (Gibco, Waltham, MA, USA); and incubated at 37 °C in 5% CO₂. The cells were grown on 100 mm dishes in 10 mL of the complete medium. The cell culture medium was changed 2–3 times per week. The cells were used after 8–10 passages. For the experiments with the investigated oil, we used DMEM without FBS and Pen/Strep. The AmO was used in the following concentrations: 0.05%, 0.1%, 0.15%. After 30 min of incubation, the cell culture media containing the studied oil were removed and the plates were washed with PBS. Subsequently, the cells were exposed to UVA radiation using a Bio-Link Crosslinker BLX 365 (Vilber Lourmat, Eberhardzell, Germany) at a dose of 10 J/cm² in 1 mL of cold PBS (4 °C). After irradiation, PBS was exchanged for fresh DMEM. The cells were incubated for 24 h. The concentration of AmO and the 10 J/cm² dose of UVA were selected based on cell viability measurements obtained from the MTT assay conducted in our previous study (data shown in previous study) [13 (link)].

Human dermal fibroblasts were obtained from the American Type Culture Collection (Manassas, VA, USA); maintained in Dulbecco’s Modified Eagle Medium (DMEM) (PANTM BIOTECH, Aidenbach, Germany); supplemented with 10% Fetal Bovine Serum (FBS) (Gibco, Waltham, MA, USA), 50 U/mL of Penicillin (Pen) (Gibco, Waltham, MA, USA) and 50 μg/mL of Streptomycin (Strep) (Gibco, Waltham, MA, USA); and incubated at 37 °C in 5% CO₂. The cells were grown on 100 mm dishes in 10 mL of complete medium. The cell culture medium was changed 2–3 times per week. The cells were used after 8–10 passages. For the experiments with the investigated oil, we used DMEM without FBS and Pen/Strep.

CxCa and OPSCC tumors were obtained and handled as described previously [5 (link), 20 (link)]. In brief, tumor material was cut into small pieces and the pieces were incubated for 60 min at 37 °C in Iscove’s Modified Dulbecco’s Medium (IMDM, Lonza, Verviers, Belgium) with 10% human AB serum (Capricorn Scientific, Esdorfergrund, Germany) and supplemented with high dose of antibiotics (50 µg/ml Gentamycin (Gibco/Thermo Fisher Scientific (TFS), Bleiswijk, the Netherlands), 25 µg/ml Fungizone (Invitrogen/TFS), 100 IU/ml penicillin (pen; Gibco/TFS) and 100 µg/ml streptomycin (strep; Gibco/TFS)). Next, the tumor pieces were put in culture in IMDM supplemented with 10% human AB serum, 100 IU/ml penicillin, 100 µg/ml streptomycin, 2 mM L-glutamin (Lonza, Breda, Netherlands; hereafter referred to as IMDM complete) and 1000 IU/ml human recombinant IL-2 (Aldesleukin, Novartis, Arnhem, the Netherlands). Cultures were replenished every 2–3 days with fresh IMDM complete and IL-2 to a final concentration of 1000 IU/ml and when sufficient T cells were obtained after 2–4 weeks the cells were harvested, cryopreserved and stored in liquid nitrogen until use.