Culturing of dendritic cells from bone marrow of FVB mice

Objective To establish a method for cultivation and amplification of mouse bone marrow-derived dendritic cells in vitro,and to observe cell morphology and related characteristics.Methods Under aseptic condition,bone marrow cells were extracted from the tibia and femur bones of FVB mice.Bone marrow mononuclear cells were isolated,and cultured in the RPMI 1640 medium with recombinant mouse granulocyte-macrophage colony-stimulating factor(rmGM-CSF) and interleukin(IL)-4 in vitro.Dendritic cells obtained were divided into two groups: lipopolysaccharide(LPS)-dendritic cell and sample dendritic cell.Dendritic cells in the LPS-dendritic cell group were treated with LPS for stimulating and the dendritic cells in the simple dendritic cell group were used as controls without treatment.The morphology of the dendritic cells was observed by inverted microscope,The biological characteristics of the dendritic cells were identified by flow cytometry.Results Two weeks after culture,the cultured cells displayed the typical morphology of dendritic cells.Under the inverted microscope,the surface of the cultured cells was irregular,and there were many dendrite-like processes on the surface.The cultured cells were indentified as myeloid dendritic cells by flow cytometry.High expressions of MHC-Ⅱ,CD80,CD86 and CD11c were observed on the surface of the cultured cell.In the simple dendritic cell group,MHC-Ⅱ expression was at a moderate level,CD80,CD86 and CD11c expressions were at a low level,and the cultured cells had the weak ability to stimulate the proliferation of T cells.In the LPS-dendritic cells group,MHC-II,CD80,CD86 and CD11c expressions were at a high level.Conclusion The method of primary culture used in this experiment can produce a large amount of dendritic cells in vitro,and the cultured cells displays the typical morphology of dendritic cells with a higher purity.