The procedure of IFA had been described previously [68 (link)]. The virus was identified using a mouse anti-VP1 pAb.
After infection, the cell suspension (2×106/mL) was prepared and stained by PI (BD). Then viable cells (no staining) were separated using flow cytometer. Next, surface expression of IFNAR1/2 was detected by flow cytometry analysis. A total of 1 × 106 viable cells were collected and washed in PBS and incubated with 2 μL of rabbit anti-IFNAR1/2 mAb and mouse anti-VP1 mAb or isotype antibody in 100 μL of PBS for 30 min at 4°C. The cells were then washed and diluted in 200 μL of PBS containingAlexa Fluor 488-conjugated goat anti-rabbit IgG (H+L) antibody (Abcam, ab96883) and Alexa Fluor 647-conjugated goat anti-mouse IgG (H+L) antibody (Abcam, ab150115) for 30 min at 4°C. The cells were washed and diluted in 500 μL of PBS and analysed using a BD Cytomics TM FC 500 instrument. FlowJo software was used for data analysis.
After infection, the cell suspension (2×106/mL) was prepared and stained by PI (BD). Then viable cells (no staining) were separated using flow cytometer. Next, surface expression of IFNAR1/2 was detected by flow cytometry analysis. A total of 1 × 106 viable cells were collected and washed in PBS and incubated with 2 μL of rabbit anti-IFNAR1/2 mAb and mouse anti-VP1 mAb or isotype antibody in 100 μL of PBS for 30 min at 4°C. The cells were then washed and diluted in 200 μL of PBS containing