Sodium pyruvate

Primary uveal melanoma cell line Mel202 was established from a uveal melanoma patient as described [49 (link)]. Mel202/DR1/CD80 vaccine cells were produced as described [7 (link), 10 (link)]. Briefly, Mel202 cells were retrovirally transduced and selected to stably express HLA-DR1 and CD80. The vaccine HLA-DR1 genotype is HLA-DRB1*0101. Cell lines were cultured at 37°C, 5% CO₂. Cells were grown in RPMI 1640 (ThermoFischer) with 1% L-Glutamin (ThermoFischer), 1% sodium pyruvate (PAN-Biotech, Germany), 1% MEM (PAN-Biotech), 0,4% MEM Vitamin Solution (choline chloride, folic acid, myo-inositol, niacinamide, D-pantothenic acid (hemicalcium), pyridoxal-HCL, riboflavin, thiamine-HCL, sodium chloride) (PAN-Biotech), 0,4% PenStrep (ThermoFischer), 0,1% mercaptoethanol (ThermoFischer) and 10% heat inactivated fetal bovine serum (FBS) (C-C-Pro, Germany). Blood samples and PBMC: Blood samples were obtained from healthy donors by leukapheresis. All cell lines and procedures with human materials were approved by the Institutional Review Boards of the participating institutions.

The human pancreatic cancer cell lines MIA PaCa-2, AsPC-1, PANC-1 and BxPC-3 were purchased from American Type Culture Collection (CRL-1420TM, CRL-1682, CRL-1469 and CRL-1687, respectively, ATCC; Rockville, MD, USA) and banked at Centre Paul Strauss. Cells were cultured at 37 °C in a humidified atmosphere containing 5% CO₂ and 95% air. PANC-1 was cultured in Dulbecco’s modified Eagle’s medium (DMEM; PAN Biotech GmbH, Aidenbach, Germany) supplemented with 10% fetal bovine serum (FBS, PAN Biotech GmbH) and 1% of a solution of penicillin (10000 IU/mL) and streptomycin (10 mg/mL) (PAN Biotech GmbH). MIA PaCa-2 was cultured in the same conditions with 1% of 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES, 1 mM, PAN Biotech GmbH), 1% of sodium pyruvate (10 mM, PAN Biotech GmbH) and 1% of non-essential amino acids (NEAA, PAN Biotech GmbH). AsPC-1 and BxPC-3 were cultured in Roswell Park Memorial Institute medium (RPMI; PAN Biotech GmbH) supplemented with 10% FBS, and 1% of a solution of penicillin (10000 IU/mL) and streptomycin (10 mg/mL). Subconfluent cell monolayers were trypsinized once a week using 0.5% trypsin containing 2% EDTA (PAN Biotech GmbH) and plated at passage ratios between 0.25:10 to 1:10, according to the cell line or used directly for study after enumeration determined with a Countess^® Cell Counter (Countess, Invitrogen, Carlsbad, CA, USA).

Equid herpesvirus type 1 strain RacL11 EHV-1-RFP [9 (link)] with a red fluorescent protein (RFP) fused to the small capsid protein VP26 [3 (link)] was used in this study. The virus further expresses the enhanced green fluorescent protein (eGFP) for efficient identification of infected cells. The virus was grown on primary equine dermal (ED) cells (CCLV-RIE 1222, Federal Research Institute for Animal Health, Germany) as described before [3 (link)]. The cells were propagated in Isocove’s Liquid Medium with stable glutamine (Pan-Biotech GmbH) supplemented with 20% fetal calf serum (Pan-Biotech GmbH), 0.5% penicillin (Roth), 0.5% streptomycin (Alfa Aesar), 1% sodium pyruvate 100 mM (Pan-Biotech GmbH), and 1% nonessential amino acids (Merck KGaA). For microscopy experiments, virus was purified by ultracentrifugation over a 30% sucrose solution followed by sucrose step gradient ultracentrifugation exactly as described before [10 (link)].