Generation of Bone Marrow Derived Murine Dendritic Cells for Use in 2-photon Imaging

Summary

Antigen presentation in secondary lymphoid organs by dendritic cells is crucial for the initiation of the T cell mediated adaptive immune response. Here we demonstrate the culture of bone marrow derived murine dendritic cells, activation, and labeling for 2-photon imaging.

Abstract

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^{1, 2}. Phase I consists of brief serial contacts between highly motile antigen specific T cells and antigen carrying dendritic cells^{1, 2}. Phase two is marked by prolonged contacts between antigen-specific T cell and antigen bearing dendritic cells^{1, 2}. Finally, phase III is characterized by T cells detaching from dendritic cells, regaining motility and beginning to divide^{1, 2}. 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.

Protocol

1) Remove both femur bones from one mouse

1. Use dissection scissors to cut away muscle and expose the femur bone above and below the joints (knee and hip). Grasp the center of the femur with dissection tweezers and cut above and below the joints to leave as much of the epiphysis intact as possible.
2. Clean off as much muscle as possible using small dissection scissors. Transfer the femur into a dish of RPMI.
3. All procedures should be carried out in the hood from this point on, using only sterile media, instruments, pipette tips and culture dishes.

2) Sterilize Femur bones:

1. In the hood, transfer bones ....

Discussion

Dendritic cells are key mediators of the adaptive immune response and the most efficient antigen presenting cell characterized to date. Methods for human and mouse dendritic cell culture in the literature differ in the type of cytokines used to influence the development of different dendritic cell types. Notably, GM-CSF, flt3L, IL4, IL13, TNF-alpha and IFN-gamma are used in different combinations to produce mature, immature, inflammatory and steady-state like murine bone marrow derived dendritic cells in vitro^3-7