The main stock (HA titer: 32768/0.05 mL, EID50: 10 9.5 /mL, protein content: 2 mg/mL) was stored as 20 µL aliquots at -80 °C till use. IAV is a Biosafety Level 2 agent and was handled following an approved biosafety protocol at Stanford University. Texas Red-DHPE was incorporated into the IAV envelope by incubating virus sample with 10 µM of Texas Red-DHPE in HB buffer at 4 °C on ice overnight such that the total EtOH (necessary for dissolving Texas Red-DHPE) concentration in the labeling mixture did not exceed 2% v/v. The labeled virus suspension was directly diluted in vesicle buffer for use in lipid mixing experiments. For content mixing experiments, the commercially available virus suspension was directly diluted in vesicle buffer.