Indicated cells were seeded 24 hours prior to infection, changed to IGM at the time of infection, and, at the indicated time points, trypsinized and resuspended in PBS supplemented with 2% of heat-inactivated fetal bovine serum (FBS). For HA staining, cells were stained with 10 μg/ml of H17-L19, a mouse monoclonal antibody. [65 (link)] For NS1 staining, rabbit polyclonal sera, Genetex catalogue GTX125990, was used at a 1:100 dilution.
For M2 staining, a mouse monoclonal antibody 14C2, from ThermoFisher Scientific was used at a concentration of 10μg/ml. Secondary antibodies were either goat anti-mouse IgG conjugated to allophycocyanin, or goat anti-mouse IgG conjugated to Alexa Fluor 405. Fixation, permeabilization and staining used BD Cytofix/Cytoperm, following manufacturers instructions.
Data processing consisted of first generating a debris gate in FlowJo prior to export to a tsv or csv and analysis using custom Python scripts, enumarated in our github repository (https://github.com/Russell-laboratory/NS1_interferon_variation ). Uninfected controls were used to set empirical gates for influenza staining and interferon reporter positivity at a 99.9% exculsion criteria. Additional gating schema described in the text, and the accompanying code.
For M2 staining, a mouse monoclonal antibody 14C2, from ThermoFisher Scientific was used at a concentration of 10μg/ml. Secondary antibodies were either goat anti-mouse IgG conjugated to allophycocyanin, or goat anti-mouse IgG conjugated to Alexa Fluor 405. Fixation, permeabilization and staining used BD Cytofix/Cytoperm, following manufacturers instructions.
Data processing consisted of first generating a debris gate in FlowJo prior to export to a tsv or csv and analysis using custom Python scripts, enumarated in our github repository (