Normal mouse serum

The expression of the F protein on the cell surface over time was evaluated by flow cytometry. CEF cells grown in a T75 flask were infected, at an MOI of 0.01, with the rHVT-GFP-F virus and maintained for 48 to 72 h before harvesting. After washing the cells twice with 5% FBS in Phosphate-buffered saline 1× (PBS 1×), non-specific antibody sites were blocked with 5% Normal Mouse Serum (catalog no. ab7486, Abcam, Cambridge, MA, USA) in FACS Buffer for 15 min at 4 °C. The cells were then incubated with anti-NDV chicken serum (#10100482, Charles River Avian Vaccine Services, Norwich, CT, USA) (1:6400) for 2 h at 4 °C. After washing twice with 5% FBS in PBS 1×, the cells were incubated with 2.5 µg/mL goat anti-chicken IgY conjugated with Alexa Fluor^® 647 (catalog no. ab150171, Abcam, Cambridge, MA, USA) for 30 min at 4 °C. The cells were then washed twice and resuspended in 5% FBS in PBS 1×. Flow cytometry was performed (Beckman Coulter Gallios, Brea, CA, USA) in a 3 laser/10 color configuration, and the data were analyzed using FlowJo software v7.6.5 (TreeStar, Ashland, OR, USA). The results are expressed as the percentage of infected cells (%). The expression level of the protein is reported as the median fluorescence intensity (MFI). For each sample, 30,000 events were acquired. The experiment was independently repeated three times.

Primary BM-MNCs were fixed with BD Cytofix for 10 minutes, washed with 0.1 M glycine, permeabilized/blocked with 0.2% Triton X-100/2% normal mouse serum (Abcam) for 1 hour, stained with mAb ASC conjugated to Alexa Fluor 647 (Santa Cruz Biotechnology catalog sc-514414) for 1 hour, and resuspended in 1 μg/mL DAPI (Sigma-Aldrich). Data were acquired with an Amnis ImageStream Mark II imaging flow cytometer and analyzed using IDEAS (Luminex Corporation). Data were analyzed as previously described (46 (link)) with modifications. Samples were run on the lowest speed, and at least 3000 images were collected with 60× objective. A masking strategy was applied to remove doublets, identify ASC speck–containing cells, and differentiate specks from diffuse staining.

To prevent non-specific binding, mononuclear cells were incubated for 10 min at 4 °C with a 1:100 dilution of normal mouse serum (Abcam, Cambridge, MA, USA, Cat. no. ab7486) prior to antibody addition. Thereafter, cells were labelled with directly conjugated monoclonal antibodies (detailed in the following paragraph). All antibodies were diluted to the optimal concentration before use. Cell staining was performed at 4 °C for 20 min. Finally, cells were filtered through a 44 μM-pore size nylon mesh (Merck, Darmstadt, Germany, Cat. no. NY4100010) and analysed by flow cytometry.