Non autologous human plasma

Monocyte-derived DCs were generated as previously described 29 . In brief, blood samples from seven healthy donors were collected after approval was granted by the responsible institutional review board (Ethikkommission der Friedrich-Alexander-Universität Erlangen-Nürnberg, Ref. no. 4158) and written informed consent was obtained. Peripheral blood mononuclear cells were isolated from whole blood using density gradient centrifugation. Monocytes were extracted from the non-adherent fraction by plastic adherence, were cultured in DC medium (RPMI; Lonza, Verviers, Belgium) containing 1% non-autologous human plasma (Sigma-Aldrich, St Louis, USA), 2 mM L‑glutamine (Lonza), and 20 mg/L gentamicin (Lonza), and were differentiated into DCs by application of 800 U/mL GM‑CSF (Miltenyi Biotec, Bergisch Gladbach, Germany) and 250 IU/mL IL‑4 (Miltenyi Biotec) on days 1, 3, and 5. After six days in culture, DCs were matured for 24 hours using a cytokine cocktail consisting of 200 IU/mL IL‑1β (CellGenix, Freiburg, Germany), 1 000 U/mL IL‑6 (CellGenix), 10 ng/mL TNF (Beromun, Boehringer Ingelheim Pharma, Germany), and 1 μg/mL PGE2 (Pfizer, Zurich, Switzerland).

Peripheral blood mononuclear cells (PBMCs) were isolated from 100 to 360 mL blood, taken from healthy donors after informed consent and approval by the institutional review board (Ethikkommission of the Friedrich-Alexander University Erlangen-Nürnberg, Ref. no. 4158), by density centrifugation with Lymphoprep (Axis-Shield PoC AS, Oslo, Norway) as described previously [37 (link)]. For the generation of moDCs, monocytes, were first separated from the non-adherent fraction (NAF) by plastic adherence, to be differentiated into immature DCs (iDCs) over the course of 6 days in DC medium, consisting of RPMI 1640 (Lonza, Verviers, Belgium) supplemented with 1% non-autologous human plasma (Sigma-Aldrich, St. Louis, MO, USA), 2 mM L-glutamine (Lonza), and 20 mg/l gentamycin (Lonza). Fresh DC medium with GM-CSF (800 IU/mL; Miltenyi Biotec, Bergisch Gladbach, Germany) and IL-4 (250 IU/mL; Miltenyi Biotec) was added on days 1, 3, and 5. On day 6, DCs were matured (mDC) with the standard cytokine cocktail consisting of 200 IU/mL IL-1β (CellGenix, Freiburg, Germany), 1000 IU/mL IL-6 (Miltenyi Biotec), 10 ng/mL TNFα (Beromun, Boehringer Ingelheim Pharma, Ingelheim am Rhein, Germany), and 1 μg/mL PGE₂ (Pfizer, Zurich, Switzerland), as described in detail previously [14 (link)]. After 24 h of maturation, DCs were used for electroporation. Cells were incubated at 37 °C with 5% CO₂.