Culture and Identification of Rat Myeloid Dendritic Cells

• 1、Department of Hepatobiliary and pancreatic surgery ,The Second Affiliated Hospital of Kunming Medical University Yunnan,kunming650101,china

Abstract：Objective: To investigate a simple and efficient access to different rat myeloid DC maturation state of culture methods. Methods: Isolated and purified from rat bone marrow mononuclear cells to 50ng / ml recombinant rat granulocyte - macrophage colony stimulating factor (GM-CSF) and 50ng / ml of interleukin (IL-4) 7 d complete culture medium ; then the tumor necrosis factor -α (TNF-α) (40ng / ml) 24 h stimulation obtained smDC, simultaneously with LPS (1μg / ml) stimulation and without stimulation for 24 h to obtain the mature DC (mDC) and immature DC (imDC). Inverted phase contrast microscopy, flow cytometry, ELISA and other testing three cell morphology, expression of cell surface markers and cytokines, while using cell counting kit to detect DC allogeneic lymphocyte activation. Results： smDC shape, size, dendritic length, ranging between the number of dendritic imDC and mDC. imDC, smDC mDC were highly expressed and express OX62, MHC-Ⅱ, CD80, CD86 of. imDC, smDC secrete cytokines lower than mDC (P <0.01), the secretion of IL-1β was no difference (P and imDC> 0.05); three caught low expression of IL-10, there was no significant difference (P> 0.05). Conclusion: It is a feasible way to induce myeloid monocytes to differentiate into imDC 、smDC and mDC using GM-CSF 、IL-4 and TNF-α in vitro.

Keywords： Dendritic cells culture in vitro Granulocyte - macrophage colony-stimulating factor identification。