Generation of bone marrow derived murine dendritic cells for use in 2-photon imaging.
Several methods for the preparation of murine dendritic cells can be found in the literature. Here, we present a method that produces greater than 85% CD11c high dendritic cells in culture that home to the draining lymph node after subcutaneous injection and present antigen to antigen specific T cells (see video). Additionally, we use Essen Instruments Incucyte to track dendritic cell maturation, where, at day 10, the morphology of the cultured cells is typical of a mature dendritic cell and <85% of cells are CD11chigh. The study of antigen presentation in peripheral lymph nodes by 2-photon imaging revealed that there are three distinct phases of dendritic cell and T cell interaction. Phase I consists of brief serial contacts between highly motile antigen specific T cells and antigen carrying dendritic cells. Phase two is marked by prolonged contacts between antigen-specific T cell and antigen bearing dendritic cells. Finally, phase III is characterized by T cells detaching from dendritic cells, regaining motility and beginning to divide. This is one example of the type of antigen-specific interactions that can be analyzed by two-photon imaging of antigen-loaded cell tracker dye-labeled dendritic cells.
Matheu
Melanie P
MP
Department of Physiology and Biophysics, University of California, Irvine, USA.
Sen
Debasish
D
Cahalan
Michael D
MD
Parker
Ian
I
D000818:Animals; D017951:Antigen Presentation; D001854:Bone Marrow Cells / Q000166:cytology*; D039521:CD11c Antigen / Q000096:biosynthesis / Q000276:immunology; D003584:Cytological Techniques / Q000379:methods*; D003713:Dendritic Cells / Q000166:cytology* / Q000276:immunology; D018984:Epitopes, T-Lymphocyte / Q000276:immunology; D051379:Mice; D036641:Microscopy, Fluorescence, Multiphoton / Q000379:methods*; D013601:T-Lymphocytes / Q000276:immunology
D039521:CD11c Antigen; D018984:Epitopes, T-Lymphocyte