4.19. BAL Analysis by Flow Cytometry

Polychromatic flow cytometry of BAL fluid samples was performed on a BD FACSAria™ instrument (Becton Dickinson Biosciences, Franklin Lakes, NJ, USA) with data acquisition and analysis performed using FACSDiva™ software. Unless stated otherwise, antibodies for these studies were obtained from BD Pharmingen and comprised MHC class II-FITC conjugate (antibody 2G9), CD11c-allophycocyanin conjugate (antibody HL-3), CD11c-phycoerythrin/Cy7 conjugate, CD3-phycoerythrin/Cy5 conjugate (antibody 145-2C11), B220 (CD45R)-phycoerythrin/Cy5 conjugate (antibody RA3-6B2), CCR3-phycoerythrin (antibody 83101, R&D Systems, Abingdon, Oxfordshire, UK). FcγR blocking agent was antibody 2.4G2. Dendritic cells were enumerated as non-autofluorescent CD3/B220⁻, MHC II⁺, CD11c⁺. Eosinophils were SSC^high, CCR3⁺, moderate CD11c⁺, low-absent MHC II, B220/CD3⁻.