F1635 25ml

Cells were fixed in 3.7% formaldehyde solution (Sigma Aldrich, Cat#F1635-25ML) in phosphate buffer saline (1:9) for 15 min at room temperature in a fume hood. The cells were then rinsed twice with fresh buffer (no fixative added). Buffer solution was replaced with 50% ethanol in distilled water for 20 min. The process was continued with 60%, 70%, 80%, 90% and 100% ethanol solutions, each left for 20 min. The final step was repeated with 100% ethanol solution left on the cells for another 20 min. After the dehydration procedure was completed, the samples were transferred from 100% ethanol into a 1:2 solution of HMDS: 100% ethanol for 20 min. The samples were then transferred to a fresh solution of 2:1 HMDS: 100% ethanol for 20 min. Finally, the samples were transferred into 100% HMDS for 20 min, and this step was repeated. When the samples were submerged in the final 100% HMDS solution, the MEAs were capped loosely in the fume hood overnight to enable evaporation for the HMDS. The samples were coated with a thin layer of gold using a sputter-coater (IN02, Emitech) at a voltage of 0.8 kV, current of 5 mA and pressure of 6 × 10⁻² mbar for 30 s. Scanning electron microscopy was performed using an EVO-SEM (EVO LS25, Zeiss) at 15 kV.

Influenza A (H3N8) containing cell culture supernatants were either incubated for 16 h at 4 °C with a final concentration of 0.1% (v/v) formaldehyde (F1635-25ML, Sigma-Aldrich, St. Louis, MO, USA) or for seven days at 37 °C with 0.05% (v/v) formaldehyde at 200 rpm. To mix the sample and to prevent virus aggregation, the solution was pipetted up and down once per day. After the procedure, the virus solution was dialyzed against PBS for 3 h at 4 °C. Dialysis tubes (ZelluTrans Roth E656.1, cutoff 3.5 kDa, width 19 mm, thickness 25 µm, Carl Roth, Karlsruhe, Germany) were soaked in PBS for at least 30 min and sealed with a clip-on top and bottom. Inactivation was confirmed as described above.

VSV, VSV*ΔG (recombinant VSV Indiana strain lacking the viral envelope protein G) and VSV-GP have been described previously [32 (link)]. VSV-GP variants containing either the wild type RSV fusion protein (F) or a codon optimized variant of RSV F (GenBank entry: EF566942) were generated by replacing luciferase with the corresponding RSV F variant in VSV-GP-Luciferase, a VSV-GP variant containing luciferase as additional transgene on position 5 [33 (link)]. Newly generated VSV-GP variants were rescued as previously described and subsequently twice plaque purified. All VSV and VSV-GP variants were produced on BHK-21 cells and concentrated via low speed overnight centrifugation through a sucrose cushion. Stocks were titrated on BHK-21 cells via tissue culture infectious dose 50% (TCID₅₀) assay. RSV and rgRSV, an RSV variant containing GFP as additional transgene, was kindly provided by M. Peeples and P. Collins (NIH, Bethesda, MD, USA). Stocks were produced and titrated as described previously [34 (link)].
FI-RSV was prepared according to the protocol for “Lot100” [20 (link)]. Briefly, RSV-containing cell culture supernatant was incubated with a final concentration of 0.025% (v/v) formaldehyde (F1635-25ML, Sigma-Aldrich) for 96 h at 37 °C. The virus was then precipitated using aluminum hydroxide.