Alexa fluor 594 conjugate

Vero cells were cultured in DMEM with 10% FBS and 1% P/S in 6 wells of 20 mm-plates with glass bottoms, and the fluorescent VLPs were added into each well. After adding the fluorescent VLPs, the lipid raft of cell membrane was labeled with the Cholera Toxin B Subunit (Recombinant), Alexa Fluor® 594 Conjugate (Thermo Fisher Scientific, US) at 5, 10, 15, 20, 30, and 60 min. The labeling was for 10 min on ice and away from light. After washing with PBS, the cells were fixed with 4% paraformaldehyde for 40 min and washed again with PBS. Then the cell nucleus was labeled with Hoechst33342 (Beyotime, China) at room temperature for 10 min and washed with PBS. In the entry inhibition assays, the lipid raft inhibitor methyl-β-cyclodextrin (m-β-CD, Aladdin reagent) was diluted to a working concentration of 10 mM using the cell culture medium and pretreated Vero cells for 10 min at room temperature before being cultured with fluorescent VLPs. The images were taken under the PE Ultra VIEW VoX double disc living cell fluorescence confocal microscope. For experiments observing the entry of VLPs in time gradients, we randomly chose 5 views and counted the number of viruses absorbing on cell membrane and the number of cells entering into cells at different time points in the inhibitor treated group and the untreated group.