Monolayers of 293T cells transiently expressing viral glycoproteins (treated overnight with 25 mU per well neuraminidase) were washed and incubated with 1% RBC suspensions (pH 7.5) for 30 min at 4°C. After the samples were rinsed to remove unbound RBCs, they were placed at 37°C for the indicated time with or without 2 mM zanamivir (pH 8.0). The plates were then rocked, and the liquid phase was collected in V-bottom tubes for measurement of released RBCs. The cells were then incubated at 4°C with 200 μl of RBC lysis solution (ammonium-chloride-potassium lysis buffer; Thermo Fisher Scientific, A1049201), where the lysis of unfused RBCs removes RBCs that have not fused with cells coexpressing envelope glycoproteins. The liquid phase was collected in V-bottom 96-well plates for measurement of bound RBCs. The cells were then lysed in 200 μl of dodecyl maltoside HEPES (DH) buffer [5 mM Hepes, 10 mM NaCl, and dodecyl maltoside (0.5 mg/ml)] 1:10 in PBS and transferred to flat-bottom 96-well plates for quantification of fused RBCs. The amount of RBCs in each of the above three compartments was determined by measuring the absorption at 405 nm.