English: APP stains gEnull viral particles. This sequence shows the 3D rendering of a z-stack of the cell shown in Figure 3 that was infected with gE null virus (green) and stained for APP (red) and DAPI (blue). Quantitative validation of infection protocol. (A) Distribution of viral particles in infected cells. Cells infected with VP26-HSV1, incubated for 7–9 hr p.i., fixed and stained for DAPI as in Figure 1. Numbers of cells in each of five categories representing distribution of viral particles as diagrammed were counted from digital images taken at random from 3 different experiments. After constitutive infection (96.2+/− 6.7% infected, top row) when virus remains in the medium throughout incubation, cells displayed GFP-labeled particles in all 5 distribution categories, including 29% that display >10 cytoplasmic particles with no evidence of nuclear GFP synthesis. These GFP particles thus must represent in-coming GFP-labeled virus. In parallel cultures infected for 1 hr with our protocol (87.5 +/− 24.4% infected, lower row), no cells were found with >10 cytoplasmic GFP-particles in the absence of nuclear GFP (boxed column), and a higher percentage of cells were found with only nuclear GFP, or both nuclear GFP and cytoplasmic GFP particles (lower row). Thus, after synchronized infection only small numbers of VP26-GFP particles are found in the cytoplasm of cells not expressing viral genes as evidenced by absence of viral-encoded VP26-GFP in the nucleus. (B) Average number of VP26-GFP labeled viral particles in the cytoplasm after synchronization. To determine the average number of incoming viral particles in the cytoplasm at later stages of infection, we counted particles at two time points (6.5–7 hr and 9 hr) after synchronized infection. At 6.5–7 hr p.i., the two predominant patterns in productively infected cells were: (1) few cytoplasmic VP26-GFP-particles whether or not there was nuclear GFP (3+/−3.0 and 3+/−2.3 cytoplasmic particles respectively); or (2) many cytoplasmic particles in cells with strong nuclear GFP (65+/−5 cytoplasmic particles). Less than 3% of cells displayed other distributions, and none had many cytoplasmic particles without nuclear GFP. At 9 hr p.i. all infected cells had strong nuclear GFP and many particles in the cytoplasm. Experiments were done in triplicate and counts (16,300 GFP particles) were made on at least three different coverslips for each condition for no less than 10 microscopic fields per coverslip.