Lsr 2 cfi

Left lungs from HD-Ad_RBD vaccinated and control mice were harvested and digested for 45 min at 37 °C in digestion buffer containing liberase 2 μg/ml and Type IV DNase I 25 units/ml. The lung infiltrated cells were cultured with purified RBD protein at 10 μg/ml for 12 h at 37 °C followed by a 6 h treatment with GolgiPlug (BD 555028). After blocking with FcγIII and FcγII receptors antibodies (BD Pharmingen, 553142), cells were stained with live/dead fixable cell stain (Invitrogen 34955), CD44 BV510, CD4 BV711, and CD8a APC-Cy™7 (BD) antibodies. Stained cells were fixed and permeabilized with Cytofix/Cytoperm Fixation/Permeabilization (BD 555028), and then intracellularly stained with anti-IFN-γ APC (BD). Cells were analysed on a Becton Dickinson LSR II CFI (SickKids Flow Cytometry Facility), using Flowjo × 10.0 software.