Inducing Culture in vitro and Phenotypic identification of Dendritic Cells from Peripheral Blood of Healthy People

Objective To isolate the PBMC from peripheral blood of healthy human,and get mature dendritic cell(DC)by inducing culture in vitro.Methods PBMCs were isolated from fresh peripheral blood of healthy people by the method of density-gradient centrifugation.The cells were cultured in RPMI-1640 supplemented with 10% fetal bovine serum at 37 ℃ in a 5% CO2 humidified atmosphere for 2 hours,and then the suspended cells were removed.Then recombinant human granulocyte-macrophage colony-stimulating factor(rhGM-CSF)and recombinant human interleukin-4(rhIL-4)were added into medium,and the adherent cells were cultured to continue for 5 d.Tumor necrosis factor-α(TNF-α)was added to promote DCs'maturation and differentiation.Then the morphological features were observed with inverted microscope and the phenotypes of cells were detected by flow cytometer(FCM).Results Under the microscope,the dendritic cells were in irregular shape with slender synapses on their surface.The expressions of CD83 and CD80 on mature dendritic cells were significantly higher on 8d than that on 4d.Conclusion Mature dendritic cells can be cultured from peripheral blood of healthy people in vitro by utilizing rhGM-CSF,rhIL-4 and TNF-α