Rpmi 1640 glutamax culture medium

Human astrocytoma cell line U251 and T cell CD4+ lymphoblastic lymphoma cell line SUPT1 were obtained from Sigma-Aldrich (Schnelldorf, Germany). Human Embryonic Kidney 293A (HEK293A) and adenocarcinoma cervix epithelial HeLa were obtained from ATCC (ATCC-LGC standards GmbH, Wesel, Germany). Human Aortic Endothelial Cells (HAEC) were obtained from Lonza and grew in EGM medium (Lonza, Basel Switzerland). HeLa and HEK293A were cultivated in DMEM medium supplemented with Streptomycin/penicillin antibiotic solution (Gibco BRL-Life Technologies, Grand Island, NY) and with 10% Foetal Bovine serum (FBS) (Gibco BRL). U251 was cultivated in RPMI-1640-GlutaMax culture medium with 1% FBS (Gibco BRL) and SUPT1 in RPMI-1640-GlutaMax culture medium with 10% FBS.

Human DCs were differentiated as described elsewhere.^{65 (link)} Briefly, magnetic sorted CD14+ cells (Sorted with CD14 Microbeads Human, MACS Miltenyi; #130-050-201) from PBMCs of healthy donors were cultured in 75 cm² cell culture flasks in RPMI 1640 GlutaMax culture medium (Invitrogen, Carlsbad, CA, USA) with 10% heat-inactivated fetal bovine serum (Invitrogen). For DCs differentiation, on day 0 and 4 the cultures were supplemented with 50 ng/mL recombinant human GM-CSF (#572904; BioLegend) and 50 ng/mL IL-4 (#574006; BioLegend). The cultures were maintained at 37°C in humidified atmosphere with 5% CO₂. For DCs maturation, on day 5 of culture, LPS (#L2630-10 MG; Sigma-Aldrich, Germany) at 100 ng/mL was added to the iDCs and the culture continued for another 48 h. The cells were harvested and used for phenotyping through staining for the surface markers CLIP (clone CerCLIP.1; #ab22606; Abcam, Cambridge, UK), MHC-II (clone Tü39; #555556; BD), CD80 (#557226; BD), CD83 (#556855; BD) and CD86 (#555660; BD) or in co-cultures with T-cells.

Human DCs were differentiated from monocytic CD14+ precursors as described previously (37 (link)). Briefly, magnetic sorted CD14+ cells from PBMCs of healthy donors were cultured in 175 cm² cell culture flasks in RPMI 1640 GlutaMax culture medium (Invitrogen, Carlsbad, CA, USA) with 10% heat-inactivated fetal bovine serum (FBS) (Invitrogen). For DC differentiation, on day 0 and 4 the cultures were supplemented with 50 ng/mL recombinant human GM-CSF and 50 ng/mL IL-4 (BioLegend). The cultures were maintained at 37°C in humidized atmosphere with 5% CO₂. For DC maturation, on day 5 of culture, LPS (Sigma-Aldrich, Germany) at 100 ng/mL was added to the iDCs and the culture continued. The cells were harvested on day 7 and used in T cell stimulation assays.

DCs were generated in vitro from magnetic-sorted CD14+cells (Miltenyi Biotec, Bergisch Gladbach, Germany) from PBMCs of healthy donors. Briefly, CD14+cells were cultured in 175 cm² cell culture flasks in RPMI 1640 GlutaMax culture medium (Invitrogen, Carlsbad, California, USA) with 10% heat-inactivated fetal bovine serum (Gibco), 1% pen/strep (Thermo Scientific), 1% sodium pyruvate (Thermo Scientific) and 1% non-essential amino acids (Thermo Scientific). For differentiation of CD14+cells into immature DCs (iDCs), on days 0 and 4 the cultures were supplemented with 50 ng/mL recombinant human GM-CSF and 50 ng/mL IL-4 (BioLegend, San Diego, California, USA). The cultures were maintained at 37°C in humidized atmosphere with 5% CO₂. For DCs maturation, on day 5 of culture, LPS (Sigma-Aldrich, Germany) at 100 ng/mL was added to the iDCs and the culture continued. The cells were harvested on day 7, loaded with peptides and used in co-cultures with autologous CD8+T cells.