Cd3ε pe cy7

BAL contents were centrifuged at 1500 × g for 10 min, and BAL fluid was removed and stored at −80 °C. BAL cells were suspended in staining media (PBS/5% PBS) containing human gamma globulin and incubated on ice for 30 min. Cells were washed and stained with antibodies that were fluorescently labeled for 30 min on ice. Streptavidin-BV605 was used to probe Mac-3-biotin. The samples which were harvested from AA + Flu and Flu-control mice were additionally stained with PB1-tetramer-PE (kindly provided by Dr. Paul Thomas, St. Jude Children’s Research Hospital). All cells were fixed with 1 × stabilizing fixative (BD Biosciences, San Jose, CA). Data were acquired using LSR Fortessa Cytometer (BD). The list of fluorochromes which were purchased from BD Biosciences which were diluted 1:50 unless indicated otherwise are as follows: (clones are indicated in parentheses) CD8α-FITC (53–6.7), CD19-PerCP/Cy5.5 (1D3), Ly6G-V450 (1A8), CD193 (CCR3)-Alexa Fluor 647 (83103), CD4-Alexa Fluor 700 (RM4–5), CD3ε-PE/Cy7 (145–2C11), CD107b (Mac-3)-Biotin (M3/84, BioLegend, 1:500). Controls included unstained cells, cells stained with single colors and matched isotypes. Data were analyzed using FlowJo v10.3 (Flowjo, LLC, Ashland, OR).