Paramagnetic bead conjugated anti cd11c

Mouse lung or spleen single cell suspension was prepared by mincing whole organs through 40 μm cell strainer (BD Falcon; Franklin Lakes, NJ) followed by red blood cell lysis (ACK lysis buffer) (Sigma-Aldrich; St. Louis, MO) for 3 min. Lung antigen presenting cells were isolated from RBC-free whole lung cells labeled with paramagnetic bead-conjugated anti-CD11c (Miltenyi Biotec; San Diego, CA) and separated using autoMACS (Miltenyi Biotec; San Diego, CA) according to manufacturer's instructions. Mouse Splenic CD4⁺ T cells were isolated from RBC-free single cells suspension from wild type naïve mice as described above; and lung CD11c⁺ mDCs were isolated from RBC-free single cells suspension from WT, C3^−/− and C3ar^−/− mice exposed to air or cigarette smoke. Mouse Splenic CD4⁺ T cells were cultured in complete media for 3 days in vitro with congenic CD11c⁺ lung mDCs (10:1 ratio, 2×10⁵ CD4⁺ T cells and 2×10⁴ mDCs) in the presence of 1μg/ml soluble anti-mouse CD3 (BD; Franklin Lakes, NJ). Milliplex kit (EMD Millipore; Billerica, MA) was used to measure concentrations of a selected group of cytokines (IL-6, IL-17) according to manufacturer's instructions.