Measurement of viral fusion was performed according to a protocol previously described (43 (link), 44 (link)). Briefly, IAV A/PR/8/34 was labeled using two fluorescent dyes, R18 (octadecyl rhodamine B chloride) and SP-DiOC18 [3,3′-dioctadecyl-5,5′-di(4-sulfophenyl)oxacarbocyanine] (Life Technologies), at a ratio of 1:2 with final concentrations of R18 of 22 µM and SP-DiOC18 of 46 µM. After intense vortexing for 60 min at RT, labeled virus was filtered through a 0.22-µm-pore-size filter. Virus was bound to cells for 30 min at a cold temperature (4°C), and after the cells were washed with PBS, the temperature was shifted to 37°C for 0, 90, or 180 min. Unfixed and unpermeabilized samples were mounted. Image analysis was carried out using the spot analysis function of ImageJ for SP-DiOC18 staining with a distinct spot size of 20 pixels and a subsequent correction for cell numbers.