Flow Cytometric Analysis of Immune Responses

A total of 1 × 10⁶ cells in BALs from immunized and challenged mice were stained with extracellular (anti-mouse CD3e-FITC, CD4-APC, CD8-APC-Cy-7, CD69-PECy-5) (eBioscience, CA, United States) markers using established procedures (32 (link)). For intracellular cytokine staining, 2 × 10⁶ splenocytes were cultured for 4 days with medium only, peptide pools (1 μg/ml of each lipopeptide) or PPD (1 μg/ml). On day 5, brefeldin A (1.5 μg/ml, 1 X; eBioscience) was added, and cultured for 5 h at 37°C and subsequently stained for extracellular markers CD3-PE Cy7, CD4-PE Cy5, CD8-APC-Cy7, and intracellular cytokines IFN-γ-PE and IL-10-FITC using our previously reported procedures. For intracellular GrB staining, 2 x 10⁶ splenocytes obtained from mice were stained for extracellular markers CD3-PE Cy7, CD49b-Alexaflour 700, CD8-APC-Cy7, and intracellular GrB-Alexaflour 647, without ex vivo stimulation, using our previously reported procedures (31 (link)). Samples were run on LSR Fortessa SORP flow cytometer and analyzed using FACS-DIVA software (Becton Dickinson, Mountain View, CA, United States). Respective isotype-matched control antibodies were used to gate non-specific staining in each experiment. Gates were set to exclude 95% of isotype control antibody stained cells in all extracellular and intracellular staining experiments.