Ifnγ apc cy7

BALF were collected by flushing the lungs with 1 ml of PBS supplemented with protease inhibitors using a winged shielded catheter (1.3630 mm, BD Utah) inserted, through an incision, in the trachea of euthanized mice. Lung cells were harvested day 5 post challenge and isolated by homogenization as previously described (Lee et al., 2017 (link)). For intracellular cytokine staining analysis of T cell responses, lung and BALF cells were stimulated with F_51–66 CD4 T cell epitope of RSV F (5μg/ml), and then the cells were fixed and permeabilized according to the manufacturer’s instructions (BD Biosciences) as described in our previous studies (Ko et al., 2015 ; Lee et al., 2017 (link)). Intracellular cytokine and surface makers for T cells were stained with antibodies for IFN-γ-APC/Cy7, IL-4-FITC, CD4-APC, CD8-PE (eBioscience/BD Biosciences). Infiltrating innate immune cells were detected with antibodies for CD11b-APC, F4/80-FITC, Ly6c-A700 or Siglec F-PE (BD Biosciences). Cellular phenotypes were collected with the Becton-Dickinson LSR-II/Fortessa flow cytometer (BD, San Diego, CA). Flow cytometry data acquired were analyzed by using Flowjo software (Tree Star Inc.).