Influenza-Specific Immune Cell Profiling

Flow cytometry was performed following the method previously described [14 (link)]. Splenocytes and lung cells acquired from the mice were individually processed to assess changes in immune cell populations. Cells were stimulated with 5 μg/mL of inactivated influenza virus for 2 h at 37 °C in complete RPMI media. Afterward, cells were washed and resuspended in stain buffer containing 0.2% bovine serum albumin (BSA) and 0.01% sodium azide in PBS for Fc-receptor blocking. Cells were incubated at 4 °C for 30 min with CD3-Cy5, CD4-FITC, CD8-PE, GL7-PE, B220-FITC, CD19-Cy7, and IgD-PE antibodies, which were purchased from BD Biosciences (San Jose, CA, USA). Afterward, cells were washed and fixed using 4% paraformaldehyde. Cells were acquired using the Accuri C6 flow cytometer and populations were analyzed by C6 Analysis Software (BD Biosciences, San Jose, CA, USA).