Illuminating the Sites of Enterovirus Replication in Living Cells by Using a Split-GFP-Tagged Viral Protein

van der Schaar, H. M.; Melia, C. E.; van Bruggen, J. A. C.; Strating, J. R. P. M.; van Geenen, M. E. D.; Koster, A. J.; Bárcena, M.; van Kuppeveld, F. J. M.

Live-cell imaging. BGM(GFPS1-10) cells were seeded in glass-bottom four-chamber 35-mm dishes (CELLview) and grown to~35% confluency before transduction with MLV mCherry-GM130 particles. Infection with CVB3-3A(S11aa2) was carried out 18 to 24 h later. Cells were washed with Fluorobrite medium (Thermo Fisher Scientific) supplemented with 8% fetal calf serum (FCS) and 25 mM HEPES just prior to imaging. Imaging was performed with a Leica SP5 confocal microscope equipped with a HyD detector and a 63ϫ (1.4-NA) oil immersion objective, and the confocal pinhole was adjusted to 95.56 m as the standard or 600 m to approximate wide-field imaging. Cells were maintained in a live-cell imaging chamber at 37°C and 5% CO 2 . GFP and mCherry were excited using a 488-nm or 561-nm laser, respectively, and the appropriate emission filter, and the positions (xyz) were marked and imaged van der Schaar et al.