Penicillin

To mimic the context of sepsis in which inflammation involves the role of endothelial cells and blood cells, we used Primary Human Umbilical Vein Endothelial cells (HUVEC) (Lonza, The Netherlands) as endothelial cells and THP-1 (ATCC, The Netherlands) as monocytes. Pooled donor-HUVEC were purchased from Lonza (C2519A, The Netherlands). Cells were cultured in EBM-2TM medium (Lonza) supplemented with EGM-2 MV SingleQuot Kit Supplements & Growth Factors (Cat. No. 3202, Lonza) and antibiotics 100 IU/ml of penicillin (Astellas Pharma, The Netherlands) and 50 μg/ml of Streptomycin (Rotexmedica GmbH, Germany). Cells were used from passage 3–7 and cultured at 37°C, 5% CO2 and saturating humidity. THP-1 cells (ATCC, The Netherlands) were cultured in Gibco TM RPMI 1640 containing L-glutamine +/HEPES + (Cat. No. 1640 52400-025) supplemented with 10% (v/v) heat-inactivated FBS (Gibco), 1%(v/v) Pen/Strep 10.000 U/ml (Gibco). THP-1 cells were kept at 37°C, 5% CO2 and saturating humidity. Cells were freshly passed twice a week to keep a density of 200.000–800.000 cells/ml and used up to passage 28.

Human umbilical vein endothelial cells (HUVEC) were isolated from umbilical cords and obtained from the Endothelial Cell Facility of the UMCG. Approval of the medical ethical committee was waived by The Medical Ethical Committee of the University Medical Center Groningen (UMCG) as umbilical cords are considered anonymous waste material. HUVEC were isolated from at least two umbilical cords to circumvent donor bias, and were cultured on 1% gelatin (Sigma, Zwijndrecht, The Netherlands) coated flasks with medium (RPMI 1640, Lonza, BE) supplemented with 20% heat inactivated FCS (Fetal Calf Serum; Thermo Scientific HyClone, Cramlington, UK), 2 mM Glutamine (Lonza, Breda, The Netherlands), 5 U/ml heparin (LEO-Pharma, Amsterdam, The Netherlands), 50 μg/ml endothelial cell growth factor, 100 μg/ml streptomycin (Rotexmedica GmbH, Trittau, Germany) and 100 IU/ml penicillin (Astellas Pharma, Meppel, The Netherlands).