Name: culture of myeloid dendritic cells from bone marrow precursors
Uploaded: 2008-07-25T18:13:00
Description: Watch this Scientific Journal Video about Culture of myeloid dendritic cells from bone marrow precursors at JoVE.com

<p class="jove_content">JB is supported by studentships from the Natural Sciences and Engineering Research Council (NSERC). Support for this project has been provided by the Canadian Institutes of Health Research, Grant MOP-67066.</p>

<p class="jove_content">This protocol has been adapted from Lutz et al.<sup>1</sup></p>
<p class="jove_title">Harvest and processing of bone marrow</p>
<ol>
<li><strong>Isolation of the tibia and femur:</strong> Euthanize the donor mouse and spray the legs with 70% ethanol. Grasp the first ankle firmly with blunt forceps and begin to cut away the skin and underlying musculature to expose the tibia. To avoid damaging the bone, cut slowly and parallel to the tibia, leaving the knee joint intact. <br /><br />To clean the femur, immobilize the knee...

<ol>
	<li>Lutz, M. B., Kukutsch, N., Ogilvie, A. L., Rossner, S., Koch, F., Romani, N., Schuler, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+advanced+culture+method+for+generating+large+quantities+of+highly+pure+dendritic+cells+from+mouse+bone+marrow.">An advanced culture method for generating large quantities of highly pure dendritic cells from mouse bone marrow.</a> <em style='font-style: italic'>J. Immunol. Methods</em>. <b>223</b>, 77-92 (1999).</li><li>Gilboa, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=DC-based+cancer+vaccines.">DC-based cancer vaccines.</a> <em style='font-style: italic'>J. Clin. Invest</em>. <b>117</b>, 1195-1203 (2007).</li></ol>

<p class="jove_content">DCs are useful for studies of innate and adaptive immune interactions, and can be employed as a vaccine vector. In this video, we have demonstrated the steps to isolate bone marrow and differentiate myeloid dendritic cells in vitro. Following the culture period, these cells can be visualized microscopically both as adherent cells, which often possess dendrites, and non-adherent round cells. The DCs can be further manipulated for antigen presentation by pulsing with antigen or stimulated for cytokine production and costim...

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culture of myeloid dendritic cells from bone marrow precursors

Myeloid dendritic cells (DCs) are frequently used to study the interactions between innate and adaptive immune mechanisms and the early response to infection. Because these are the most potent antigen presenting cells, DCs are being increasingly used as a vaccine vector to study the induction of antigen-specific immune responses. In this video, we demonstrate the procedure for harvesting tibias and femurs from a donor mouse, processing the bone marrow and differentiating DCs in vitro. The properties of DCs change following stimulation: immature dendritic cells are potent phagocytes, whereas mature DCs are capable of antigen presentation and interaction with CD4+ and CD8+ T cells. This change in functional activity corresponds with the upregulation of cell surface markers and cytokine production. Many agents can be used to mature DCs, including cytokines and toll-like receptor ligands. In this video, we demonstrate flow cytometric comparisons of expression of two co-stimulatory molecules, CD86 and CD40, and the cytokine, IL-12, following overnight stimulation with CpG or mock treatment. After differentiation, DCs can be further manipulated for use as a vaccine vector or to generate antigen-specific immune responses by in vitro pulsing using peptides or proteins, or transduced using recombinant viral vectors. 

Watch this Scientific Journal Video about Culture of myeloid dendritic cells from bone marrow precursors at JoVE.com

Culture of myeloid dendritic cells from bone marrow precursors

<p class="jove_content">This video demonstrates the procedure for differentiating myeloid dendritic cells from mouse bone marrow. Isolation of mouse tibia and femur, and processing of bone marrow are demonstrated. Pictures demonstrating cell morphology before and after differentiation, and figures depicting cell phenotype and IL-12 production following maturation using CpG are shown.</p>

Myeloid dendritic cells (DCs) are frequently used to study the interactions between innate and adaptive immune mechanisms and the early response to ...

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