Flow cytometry analysis
Lung tissue was harvested at days 0, 3, 5, 7 and 21 days post-infection
(dpi). These were prepared as single-cell preparations suitable for flow
cytometry analysis by collagenase and DNase digest as described25.
Selected cell subpopulations were identified using an optimised panel of
fluorescently conjugated monoclonal antibodies (Biolegend, BD
Pharmingen) directed against cell-surface antigen markers (including
I-A/I-E, F480, Ly6G/C, B220, CD11c, CD103) as described in supplementary
Figure S1. A pooled cell preparation for each biological replicate was
used to assess background fluorescence (non-stained control) and
non-specific staining (fluorescence minus one control). Data from all
stained and non-stained tissues was acquired on a BD FACS Canto (Becton
Dickinson, New Jersey, USA), with RFP signal from fluorescently tagged
RSV also collected. Cell subpopulation analysis and RSV co-localisation
was completed used FlowJo software (Tree star, Ashland, OR), with gating
strategies presented in Figure S1.