Expression of CD80 by HSV-CD80 in infected DCs.

<p>A<u>) Detection of HSV-1 gC in infected DCs</u>. Subconfluent monolayers of DCs isolated from C57BL/6 mice were infected with 1 PFU/cell of HSV-CD80, parental virus, or mock infected. At 24 hr PI, cells were harvested and reacted with anti-CD11c and anti-HSV-1 gC antibodies and FACS analysis was performed (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0087617#s4" target="_blank">Materials and Methods</a>). Experiments were repeated twice; <u>B) FACS analyses of infected DCs for expression of HSV-1 gC and CD80</u>. Monolayers of DCs were treated as above. At 24 hr PI, cells were harvested and reacted with anti-CD11c, anti-HSV-1 gC, and anti-CD80 antibodies and FACS analysis was performed. CD11c⁺ cells were gated for expression of HSV-1 gC and CD80. Experiments were repeated twice; <u>C) Immunostaining of HSV-CD80 infected DCs</u>. DCs were infected with 1 or 10 PFU/cell of HSV-CD80 or parental virus. At 12 and 24 hr PI, cells were stained with anti-HSV-1 gC antibody and subjected to IHC as descried in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0087617#s4" target="_blank">Materials and Methods</a>; and <u>D) Quantification of photomicrographs from C</u>. Different areas of 3 slides/virus/pfu/time point from IHC described above were imaged and the number of HSV-1 gC⁺ cells was counted. Each point represents the mean ± SEM of HSV-1 gC⁺ DCs from 12 images.