2.11 Culture of langerhans cells (LC) from PBMC
PBMCs were isolated from EDTA-anticoagulated fresh blood by Ficoll density gradient centrifugation (Ficoll, Sigma-Aldrich, German). After several washes with PBS, 1*106 PBMC with culture medium(RPMI-1640 with 10 % FBS) were planted in 24-well plates. Standing at room temperature for 35 min, the supernatant and suspended cells were gently removed. Then cells were cultured in complete medium containing 100ng/ml GM-CSF,20ng/ml rhIL-4,20ng/ml rh TGF-β1 for 6 days. On the 2nd and 4th days, the medium was changed half, and on the 6th day, the cells were collected for flow cytometry. APC Mouse Anti-Human CD1a and PE anti-human CD207 (Langerin) Antibody were used to flow cytometry analysis, and the method was described previously.