Fitc conjugated cd11c

BMDCs were stimulated with Rv2005c (10 μg/mL) for 24 h. The stimulated cells were harvested and washed with PBS. The BMDCs were stained with PE-conjugated anti-CD40, anti-CD80, anti-CD86, anti-MHC class I and anti-MHC class II and with FITC-conjugated CD11c (eBioscience) for 30 min at 4 °C. The cells were washed with PBS and suspended in 250 μL PBS. The fluorescence was measured by flow cytometry, and the data were processed using FlowJo software.

To obtain lung cell suspensions, we removed and minced lungs from mice. The minced materials were incubated in complete RPMI and collagenase A (Roche, Indianapolis, IN) for 1 h at 37 °C and digests were filtered through a nylon mesh (40 μm). Dissociated cells were centrifuged at 1300 rpm for 10 min at 4 °C, and the cell pellet was incubated for 5 min in room air with ACK lysing buffer (Life Technologies, Grand Island, NY) to lyse RBCs. The single cell suspension was labeled with FITC-conjugated CD11c (eBioscience) and PE-conjugated F4/80 (Biolegend). AMs were sorted based on their expression of CD11c and F4/8042 by FACS Aria (BD Biosciences, San Jose, CA).