Time lapse video microscopy of egress was performed as previously described[9 (link)]. Briefly, tightly synchronized schizonts were percoll-enriched and arrested with 1 μM C2 for 4 h. After C2 wash out, DIC and mCherry images were collected every 5 and 25 s, respectively, for 30 min using a Nikon Eclipse Ni-E wide field microscope fitted with a Hamamatsu C11440 digital camera and a Nikon N Plan Apo λ 100x/1.45NA oil immersion objective. For each experiment, videos of the RAP- and DMSO-treated parasites were taken alternately to ensure that possible differences in the rate of egress were not a result of variation in the maturity of the parasite populations. The images were then annotated using Axiovision 3.1 software and exported as AVI movie or TIFF files. Individual egress events were annotated by detailed visual analysis of the movies, and the delay to the time of egress was recorded for each schizont for subsequent statistical analysis. Mean fluorescence intensity values of individual mCherry-expressing schizonts right before and after PVM breakdown were determined from exported raw image files (TIFF format) as described previously[9 (link)] and using the elliptical selection tool and ‘Histogram’ options of ImageJ/Fiji V1.0. DPAP3KO parasites were analyzed to determine the residual background fluorescence derived from the hemozoin.
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