IAV infected and uninfected mDCs have similar CMV antigen load.

<p>(<b>A</b>) mDCs were exposed to infectious IAV, HI IAV or untreated for 4 hr, washed and exposed to increasing doses of whole, γ-irradiated CMV for an additional 3 hr. DCs were then washed, adhered to coverslips and fixed with PFA. After permeabilization, samples were stained for CMV pp65 (green), IAV (red) and HLA-DR (blue). The entire volume of each cell was analyzed using confocal microscopy (100× 1.47NA oil objective, 7× digital zoom) and one single optical slice is shown through the center of the cell with arrowheads pointing to CMV pp65+ structures. Scale bar 5 µm. (<b>B</b>) The frequency of CMV pp65+ uninfected mDCs (blue), IAV infected mDCs (black) or HI IAV stimulated mDCs (white) after 3 hr of CMV exposure was determined by analyzing entire z-stacks of DCs and counting the number of cells that had virus associated with them. The graph shows average frequency of CMV positive DCs ± SD, with 100 cells analyzed per donor and condition (n = 3). (<b>C</b>) The average number of CMV pp65+ puncta per cell after 3 hr of CMV exposure was determined by analyzing entire z-stacks of DCs and counting the number of green (CMV) puncta per nucleated cell, assessed by DAPI staining. The graph shows average frequency of CMV positive DCs ± SD, with 100 cells analyzed per donor and condition (n = 3).