Culturing Primary Human Cells for HCMV Infection

Primary human foreskin fibroblasts (HFFs, ATCC SCRC-1041™) and human embryo kidney 293 cells (HEK 293) (Microbix Biosystems Inc., Mississauga, ON, Canada) were cultured in Dulbecco’s modified Eagle’s medium (DMEM), which was supplemented with 10% fetal calf serum (FCS) (Sigma-Aldrich, Milan, Italy), as we previously described [15 (link)]. The HCMVs that were used in this study were all bacterial artificial chromosome (BAC) clones. The clones of the endotheliotropic HCMV strain TB40/E wild-type and a mutant virus that is unable to express UL83-encoded pp65 (v65Stop) have been described previously [16 (link)]. The viruses were propagated on HFFs and were titrated by standard plaque assay [15 (link)]. HCMV infections were all performed at a multiplicity of infection (MOI) of 1. UV-inactivated HCMV were prepared using a double pulse of UV-B light (1.2 J/cm²). The UV-inactivated HCMV did not replicate or produce detectable levels of immediate-early (IE) gene products.