Egm 2 endothelial growth singlequot kit supplement growth factors

Primary human lung microvascular endothelial cells (HMVEC-L/HLMVEC) (cat: #CC-2527, Lonza, Breda, The Netherlands) and Human umbilical vein endothelial cells (HUVEC) (cat: #CC2519, Lonza) were cultured in EBM-2 supplemented with EGM-2 endothelial growth SingleQuot kit supplement & growth factors (Lonza). All experiments were performed using passage 6 for HMVEC-L and passage 5 for HUVECs. The African green monkey Vero E6 cell line (ATCC CRL-1586) was cultured in Dulbecco’s minimal essential medium (DMEM) (Thermo Fisher Scientific, Waltham, MA, U.S.A), high glucose supplemented with 10% fetal bovine serum (FBS) (Life Science Production), 1% penicillin (100 U/mL), and 1% streptomycin (100 U/mL) (Gibco- Thermo Fisher Scientific). All cells were maintained at 37°C under 5% CO2 conditions.

Human adenocarcinoma active furin-deficient LoVo cells were cultured in Ham’s medium (Invitrogen) supplemented with 20% FBS. Aedes albopictus C6/36 cells were cultured in MEM (Life Technologies) supplemented with 10% FBS, HEPES (25 mM), sodium bicarbonate (7.5%), penicillin (100 U/mL), streptomycin, glutamine (100 μg/mL) and non-essential amino acids. Baby hamster kidney cells clone 15 (BHK-15) were maintained in DMEM supplemented with 10% FBS, penicillin (100 U/mL), streptomycin (100 μg/mL), non-essential amino acids (100 μM) and HEPES (10 mM). HEK-Blue hTLR2 and HEK-Blue Null1 cells (InvivoGen) were cultured in DMEM supplemented with 10% FBS, penicillin (100 U/mL), streptomycin (100 μg/mL) and maintained according to the manufacturer’s instructions. Buffy coats obtained from anonymized, DENV-seronegative, Sanquin blood donors were used to isolate PBMCs. The PBMCs were cryopreserved at -196 °C. Primary human umbilical vein endothelial cells (HUVEC) (Lonza, the Netherlands) were cultured in EBM-2 supplemented with EGM-2 endothelial growth SingleQuot kit supplement & growth factors (Lonza, the Netherlands). All of the cell lines used in this study tested negative for the presence of Mycoplasma spp. using a commercial functional method (Lonza, the Netherlands) and/or in-house qPCR assay adapted from Baronti et al [64 (link)].