Purification of DC from tissues and generation of bone marrow derived dendritic cells

Spleen (SP), lymph node (LN), thymus and lungs were digested with collagenase D (0.5mg/ml) and DNAse (25U/ml) in 5% FCS RPMI media at 37°C for one hour. Tissues were mechanically disaggregated. Shaved skin was removed and incubated in medium with dispase (25 mg/ml) at 37°C for 1–1.5 hours. Epidermis was peeled from the dermis and incubated in medium with 2.5% Trypsin, 1mM Ethylenediaminetetra-acetic acid (EDTA) and DNAse (25 U/ml) at 37°C for 1.5 to 2 hours. The dermis was incubated in medium with collagenase D (0.5 mg/ml) and DNAse (25 U/ml) at 37°C for 1 hour. Cell suspensions were used for staining and analysis of DC. CD11c+ DC from LN and SP were purified by using anti-CD11c antibody coupled MACS beads according to the manufacturer’s instruction (Miltenyi Biotec., Auburn, CA). For in vitro stimulation of DC, CD11c+ DC were cultured for 24 hours in the presence or absence of 0.1μg/ml Lipopolysaccharide [(LPS) Sigma, St. Louis, MO].

Bone marrow derived dendritic cells (BMDC) were generated from bone marrows of WT C57BL/6 and CD5^-/- mice as previously described [38]. Briefly, bone marrow cells were extracted from the femurs and tibias and cultured in 10% Fetal Calf Serum (FCS) RPMI1640 media supplemented with 10 ng/ml of recombinant Granulocyte-macrophage colony-stimulating factor [(GM-CSF) BD Bioscience] and 10 ng/ml IL-4 (Sigma) at 1x10⁶cells/ml. LPS (1 μg/ml) was added to stimulate maturation of BMDC at day 5 and BMDC were harvested 24 hours later. Around 95% cells expressed DC marker CD11c.