An infectious recombinant equine arteritis virus expressing green fluorescent protein from its replicase gene, by E. van den Born, C. C. Posthuma, K. Knoops and E. J. Snijder
Journal of General Virology vol. 88, part 4, pp. 1196 – 1205
Supplementary material. In vivo imaging analysis of Vero-E6 cells infected with EAV-GFP2
Vero-E6 cells were grown to 70 % confluence in a glass-bottomed 10 cm2 culture dish (MatTek Corporation) and were infected with EAV-GFP2 at an m.o.i. of 10. After 1 h, the inoculum was removed and 5 ml prewarmed Dulbecco's modified Eagle's medium containing 2 % fetal calf serum was added. The dish was placed in the 37 °C observation chamber (containing 5 % CO2) of a Leica TCS SP5 confocal microscope operating under the Leica Application Suite Advanced Fluorescence software. From 3 to 18 h p.i., a z stack of five images (total thickness, 6.5 µm) was recorded every 5 min with the pinhole set at 3 Airy units. A 488 nm laser was used for eGFP excitation and emission was recorded at 500–650 nm. Subsequently, movie frames were created by merging the five images of the z series captured at each time point and were processed into a movie of six frames s−1 by using QuickTime Pro.
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