To estimate the number of HSV-1 genomes expressed in each infected cell, we obtained images (as described above) using a Nikon Eclipse Ti-E epifluorescence inverted microscope. Each experimental condition (different cells, viruses, inhibitors, and MOI) was replicated in two wells, and the experiment was performed at least twice. From an individual well, five random areas were imaged. From each image, 100 cells were analyzed for their color content. To define the average number of incoming genomes being expressed, we used the mathematical equation for estimating the most likely average number of genomes expressed in each cell (λ) according to the number of one- (r1), two- (r2), or three-color (r3) cells out of the number of cells analyzed (n), as was previously developed (Kobiler et al., 2010 (link)):
The λ was calculated for each well individually (based on 500 cells) and for each condition the mean λ and standard deviations were calculated. To predict the significance of the difference between the condition a two tailed student T-test was performed.
The λ was calculated for each well individually (based on 500 cells) and for each condition the mean λ and standard deviations were calculated. To predict the significance of the difference between the condition a two tailed student T-test was performed.
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