Analyzing Immune Cell Dynamics in Spleen and Lung

The activity of immune cells in the spleen and lung was analyzed by flow cytometry as described previously [21 (link)]. Percentages of T cells (CD4⁺, CD8⁺) and B cell (germinal center) from splenocytes and lung cells of mice were analyzed by flow cytometry 4 days post-challenge as described previously [6 ,22 (link)]. Splenocytes and lung cells (1×10⁶ cell/mL) in staining buffer (2% bovine serum albumin and 0.1% sodium azide in 0.1 M PBS) were incubated at 4°C for 15 min with Fc Block (BD Biosciences, CA, USA). For surface antigen staining, cells were incubated with fluorophore-conjugated antibodies (CD3e, CD4, CD8, B220, GL7; BD Biosciences) at 4°C for 30 min. Splenocytes and lung cells were washed with staining buffer and fixed with 4% paraformaldehyde for 30 min at 4°C before acquisition using a BD Accuri C6 Flow Cytometer (BD Biosciences). Data were analyzed using C6 Analysis software (BD Biosciences).