Tnf α

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.

Human umbilical endothelial cells (HUVEC) were purchased from Lonza (Breda, The Netherlands) and cultured in EBM-2 medium supplemented with EGM-2 MV SingleQuot Kit Supplements and Growth Factors (Cat No. CC-3202, Lonza) at 37 ℃ in an incubator containing 5% CO₂. All cell cultures were performed and maintained by the UMCG Endothelial Cell Facility. HUVEC from passages 5 to 7 were seeded on culture plates (Costar, Corning, USA) with various cell densities to obtain 50–90% confluency as indicated.
Where indicated, HUVEC were activated with 10 ng/mL of TNF-α (Boehringer, Ingelheim, Germany) for 4 h prior to adding LNP samples. uLN and AbLN containing RelA specific siRNA were added and incubated with the activated cells for 24 h at the indicated siRNA concentrations. TNF-α was present during the LNP incubation period. Subsequently, cells were washed and cultured for an additional 24 h in fresh medium without LNP and TNF-α. Additionally, at the 44 h time point, cells were re-challenged with LPS (1 μg/mL) for 4 h and next harvested for gene and/or protein expression analysis after being washed with phosphate-buffered saline (PBS). Lipofectamine-mediated siRNA_RelA transfection as control was also performed in the same activated HUVEC model, according to the manufacturer’s protocol.