Antibiotic antimytotic aa

Primary cultures of human monocyte-derived dendritic cells were generated from peripheral blood monocytes (PBMCs) obtained from buffy coats of healthy individuals (informed consent obtained and approved by RadboudUMC ethical committee) according to institutional guidelines and as described previously (de Vries et al., 2005 (link)). Monocytes were differentiated into dendritic cells by culturing for 6 days at 37°C under 5% CO₂ in complete RPMI-1640 (Gibco, ThermoFisher) supplemented with 10% fetal bovine serum, 2 mM UltraGlutamine (Lonza), 1% antibiotic-antimytotic (AA, Gibco, ThermoFisher), 300 U/ml IL-4 and 450 U/ml granulocyte-macrophage colony-stimulating factor (GM-CSF).

RAW 246.7 cells were cultured in RPMI-1640 medium (Gibco) supplemented with 10% Fetal Bovine Serum (FBS, Greiner Bio-one), 1mM Ultra-glutamine (BioWitthaker) and 0.5% Antibiotic-Antimytotic (AA, Gibco). iDCs were derived from PBMCs as described previously (32 (link), 33 (link)) and cultured in RPMI 1640 medium (Gibco) supplied with 10% Fetal Bovine Serum (FBS, Greiner Bio-one). Transfections with t-Epac-vv (34 (link)) (gift from K. Jalink), Gα_s-GFP (gift from M. Rasenick), Gα_i-GFP and Gα_i1-Citrine (35 (link)) (gift from A. Gilman), Gγ₂-CFP and Gβ₁ wildtype (both gifts from M. Adjobo-Hermans) were performed with Fugene HD (Roche) according to the manufacturer protocol and imaged after 24 h. Stable cell lines expressing Gα_s-GFP and Gα_i-GFP was maintained using the appropriate antibiotics. Cells were plated one day prior to measurements or transfection in Willco dishes (Willco Wells BV) at 400,000 cells/dish or in 96 well-plate (microplate BD Falcon) at 40,000 cells/well or in 4-well Lab-Tek II chambered coverglass (Nunc) at 100,000 cells/chamber. Prior to imaging, the medium was replaced with 1 ml RPMI medium without phenol red to avoid background fluorescence.