Characterization of microglial-like cells. Monocytes were isolated from peripheral blood mononuclear cells (PBMCs) of healthy donors, and then cultured either in absence or presence of astrocyte-conditioned medium (ACM). (A) Cell morphology was observed by phase-contrast microscopy. Monocytes cultured in the absence of ACM were used as control cells. The results shown are representative of at least three different donors. (B) ACM-differentiated cells were permeabilized and stained with anti-ionized calcium binding adaptor protein-1 (IBA1) antibodies. IBA1 expression was observed by epifluorescence microscopy. The results shown are representative of at least three distinct donors. (C) Substance P secretion by monocyte-derived microglial-like cells (MDMis) and non-ACM-treated control cells was evaluated by slot-blot. Results from two independent donors (means ± SEM) are presented. (D) ACM-treated and non-treated monocytes were infected with YU2. Virus replication was assessed by monitoring the p24 content at days 3, 6 and 9 post infection. The means ± SEM are calculated from three independent experiments with triplicate samples. (E) The surface expression of BLT1 and cysLT2 receptors on ACM-treated monocytes was determined by flow cytometry. The results shown represent a single donor out of a total of four.