Temperature stress test to evaluate stability of pre-F conformation on RSV-infected HEp-2 or A549 cells.

HEp-2 and A549 cells were cultured to confluence as described above and infected with RSV at an MOI of 0.07. At 72 h postinoculation, the infected and uninfected cells were washed with PBS and detached with Versene solution for 20 min at 36°C (0.48 mM EDTA in PBS; Thermo Fisher; catalog number A4239101). Next, the cells were resuspended in PBS at 10⁶ cells/400-μL aliquot and heated at various temperatures (36°C, 45°C, 50°C, 55°C, 60°C, and 65°C) for 10 min in a digital heat block. Cells were kept at 4°C until ready to stain with the same protocol as described above. After fixation, cells were analyzed on Amnis Mark II ImageStream (405-nm, 488-nm, 561-nm, and 642-nm excitation lasers; magnification, ×40; EMD-Millipore) and data analysis was performed using IDEAS software (Amnis).