Analyzing Influenza Virus Infection in B Cells

B cells from normal mice were infected with LAIV or incubated with inactivated WSN (BPL treatment) at an MOI of 1 in RPMI 1640 medium containing 0.5 μg/ml TPCK-trypsin and incubated at 37 °C for 1 h. The infected cells were washed five times with PBS and labeled with CFSE (Merck) (1 μM CFSE for 10 min at room temperature, then washed twice with complete RPMI 1640 plus 10% FCS), then incubated with GM-CSF-cultured bone marrow-derived dendritic cells (BMDC) (ratio: 1:3) in DC culture medium (RPMI 1640 supplemented with 20 ng/mL murine GM-CSF (R&D Systems), 10% FBS, 50 μM 2-ME, 100 units/mL penicillin, and 100 μg/mL streptomycin) for 24 h at 37 °C. After incubation, DCs were isolated by M-pluriBead Cell Separation kit (pluriSelect) following the procedure from the company. The number of DCs with the green fluorescent signal was analyzed by flow cytometry, and viral proteins were analyzed by western blot^{41 (link)}. Some isolated DCs were incubated in DC culture medium for another 24 h at 37 °C for detection of virus replication.