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The Investigation of Bone Marrow-derived Dendritic Cell by Atomic Force Microscopy

Author: WangJiongKun
Tutor: XingFeiYue
School: Jinan University
Course: Immunology
Keywords: Atomic force microscopy Bone marrow cells Dendritic cells Ultrastructure Roughness Adhesive forces
CLC: R392
Type: Master's thesis
Year: 2009
Downloads: 47
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Abstract


Objective:To investigate the ultrastructure and mechanical properties of cellular membrane of granulocyte-macrophage colony stimulating factor(GM-CSF) induced bone marrow-derived immature and mature dendritic cell(DC) by atomic force microscopy(AFM).Methods:A model of bone marrow-derived DC induced by GM-CSF in vitro was established,and the changes on the morphology of bone marrow cells were observed by optical microscopy and AFM.CD11c positive DCs were selected by magnetic activated cell sorting technique(MACS) and the selected DCs were divided into three group:control,Jagged-1 group and LPS group.DCs in each group were then labeled with fluorescin-conjugated monoclonal antibody and MHC II expression on these cells was evaluated by flow cytometry.The supernatants were collected and detected by ELISA for the production of IL-12.The morphology of immature and mature DC was observed by both SEM and AFM.The cell size,roughness and adhesive force of cellular membrane was quantitatively analyzed by AFM.Results:In day 6,there is obvious differentiation occurred in the GM-CSF induced bone marrow cells,mainly including the oval or round semi-adherent cells in aggregates and the shuttle-like adherent cells in dispersion.After treatment with LPS,the percentage of MHC II positive DCs enhanced greatly and IL-12 level was significantly high than that in control and Jagged-1 group, suggesting that LPS has promoted DC functional maturation.The observations by SEM confirmed that DC in control and Jagged-1 group was smaller with little and short processes,while DC in LPS group was bigger with abundant and long processes.The morphology of immature and mature DC obtained by AFM was consistent with that by SEM,but there is little shrinkage occurred in the AFM imaged DC’ membrane and processes,suggesting that less damage to the ultrustructure was made by AFM.AFM quantitative analysis revealed that both roughness and adhesive forces on the surface of mature DC were higher than that of immature DC.Conclusion:By contrast with SEM, AFM causes less damage to cell,which is facilitate for observing ultrastucture of cellular membrane clearly and finely.Thus,AFM provides a powerful tool for investigating cells morphologically similar to DC in term of cellular process.The roughness and adhesive forces of mature DC’ membrane are higher than that of immature DC,suggesting that the reorganization of cytoskeleton during DC’ differentiation has great influence on the ultrustructure and mechanical properties of DC’ membrane,providing new evidences for DC’ maturation.

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