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.
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 interaction1, 2. Phase I consists of brief serial contacts between highly motile antigen specific T cells and antigen carrying dendritic cells1, 2. Phase two is marked by prolonged contacts between antigen-specific T cell and antigen bearing dendritic cells1, 2. Finally, phase III is characterized by T cells detaching from dendritic cells, regaining motility and beginning to divide1, 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.
1) Remove both femur bones from one mouse
2) Sterilize Femur bones:
3) Collect Bone marrow under sterile conditions:
4) Re-suspend and Spin down Bone Marrow cells:
5) Cell Lysis:
6) Count Cells:
7) Plating Dendritic cells:
8) Culture Care and Maturation:
Always check cultures before adding more media or using in any experiments. Check the general health of cells and look for potential contamination.
DAY 0: Dendritic cells plated.
DAY 3: Remove 75% of the media and non-adherant cells and add back Primary DC media.
DAY 6: After Initial Cell Plating: Re-plate the cells using the following procedure:
DAY 10-11
Mature DCs ready for stimulation/antigen loading.
9) Dendritic Cell Stimulation:
Dendritic Cell Media: Sterile filter after everything has been added
Please see the discussion section for a review of different dendritic cell culture conditions and for the resulting dendritic cell phenotype. These culture conditions are designed to produce mature dendritic cells that rapidly home to draining lymph nodes, 18-24 hours after subcutaneous injection and present antigen efficiently.
Primary DC Media: Sterile filter after everything has been added
SecondaryDC media for maturation*
100 ng/mL TNF-alpha (add to Primary DC media)
* Note: GM-CSF + IL-4 should produce immature dendritic cells. Using GM-CSF + IL4 (400 IU/ul) + TNF-alpha (100 ug/mL) will create cells that resemble mature monocyte derived dendritic cells. Mature dendritic cells are known not to take up antigen as efficiently as immature dendritic cells and cultures may have endogenous TNF-a released from stromal cells4.
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 vitro3-7. Here, we present a simple method for the production of mature murine dendritic cells that are capable of homing to draining lymph nodes after subcutaneous injection, presenting antigen and activating na ve T cells. The resulting dendritic cell population is typically >85% CD11chigh with inducible expression of CD80/86 upon LPS activation. The addition of TNF-alpha to the culture on day 7 is optional and produces a more mature phenotype8. The maintenance of in vitro Langerhans dendritic cell cultures, TNF-alpha is an important survival factor but does not stimulate the cells to mature9. It should be noted that TNF-alpha from endogenous sources (likely stromal cells) has been reported to be present in the culture media7. Other successful methods for imaging subcutaneously injected dendritic cells include positive selection for CD11c+ cells from the spleen as well as concurrent labeling and activation of the endogenous dendritic cell population1, 2, 10. Production of CD11chigh dendritic cells from bone marrow is a robust method that consistently produces large numbers of dendritic cells capable of antigen presentation in vivo, important in imaging the process of adaptive immune system activation11, 12.
National Institutes of Health Kirchstein Fellowship predoctoral fellowship AI-64128 (MPM), GM-41514 (MDC), GM-48071 (I.P.)