Figure 6

The fusion inhibitor T20 moderately enhances target CD4 positive BeWo cells-HIV-1/LAI infected PBMCs fusion. Target CD4 positive BeWo cells (2 × 10⁴/well) were coated overnight on slides as described in the method section. Target cells were then pre-incubated with indicated doses of AMD3100 or T20, or without drug, for 30 minutes at 37°C. At this point, calcein-AM loaded, HIV-1 LAI infected PBMCs were added ant left to interact for 5 h at 37°C. Slides were then washed and the cells fixed. The nuclear dye DAPI was then added into the wells for 5 min and washed out afterwards. The slides, once dried, were mounted in the presence of an antifade reagent at left to cure overnight at +4°C, in the dark. The day after, slides were observed under fluorescent microscope (20× magnification) and the images captured. Phase contrast images were also captured. The figure shows representative microscope fields of 2 (no drug or AMD3100) or 4 (T20) different wells for each experimental conditions tested. Row 1: Calcein stain; row 2: DAPI stain; row 3: merge of 1 and 2; row 4: phase contrast images. The green spots and green areas are intact PBMCs and their fused cytoplasm with BeWo cells, respectively. The blue spots are cell nuclei coloured by DAPI. The figure shows moderate enhancement of viral passage by T20 on CD4 positive BeWo cells.