Imaging and Quantifying Shed Virions

Infected cells (MOI ≈ 0.003) were imaged with a Nikon Ti2 confocal microscopy system using a 40×, 1.30-NA objective at 16 h.p.i. Infected cells were selected based on surface expression of HA (using 9 nM CR9114 Fab for H1N1 strains and 19 nM FI6v3 plus 19 nM H3v-47 [71 (link)] for H3N2 strains) and M2 (using a Fab fragment derived from mAb148 [72 (link)]). Confocal z-stacks spanning a range of 15 μm were collected for the channel corresponding to labeled HA. Image analysis was performed on Nikon NIS Element software 5.21. Briefly, a maximum intensity projection was generated from confocal z-stacks, from which the body of the infected cell was identified by size and intensity and stored (Mask A). This mask was dilated by ~1 cell diameter to capture a concentric region surrounding the infected cell (Mask B). Peak detection was performed within the region between Masks A and B, where bright spots corresponding to shed virions were identified and counted.