This study was funded by the Deutsche Forschungsgemeinschaft to S.W. (DFG WA 2804) and a university grant to S.W. from the Experimental and Clinical Research Center, a cooperation of the Max Delbr&uuml;ck Center for Molecular Medicine and Charit&eacute; Medical Faculty in Berlin. The funders had no role in study design, data collection and analysis, decision to publish or preparation of the manuscript. We thank Mr. Robert Westphal for technical support during his internship in our laboratory.

All animal procedures must be approved by the local institutional animal welfare committee prior to execution. During the MR measurements an adequate level of anesthesia and physiological monitoring (body temperature, respiratory rate) are indispensable requirements.
1. Generation of Mouse Bone Marrow-derived Dendritic Cells
<ol>
	<li>Extract bone marrow cells from C57BL/6 mice as previously described 12. This protocol dates back to 1992 13 and was originally des...

<ol>
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(17), e773 (2008).</li><li>Inaba, K., Inaba, M., Romani, N., Aya, H., Deguchi, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Generation+of+large+numbers+of+dendritic+cells+from+mouse+bone+marrow+cultures+supplemented+with+granulocyte/macrophage+colony-stimulating+factor.">Generation of large numbers of dendritic cells from mouse bone marrow cultures supplemented with granulocyte/macrophage colony-stimulating factor.</a> Journal of Experimental Medicine. 176 (6), 1693-1702 (1992).</li><li>Lanzavecchia, A., Sallusto, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ralph+M.+Steinman.+1943-2011.">Ralph M. Steinman. 1943-2011.</a> Cell. 147 (6), 1216-1217 (1943).</li><li>Srinivas, M., Turner, M. S., Janjic, J. M., Morel, P. A., Laidlaw, D. 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E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+signal-to-noise+ratio+of+the+nuclear+magnetic+resonance+experiment.">The signal-to-noise ratio of the nuclear magnetic resonance experiment.</a> J. Magn. Reson. 24 (1), 71-85 (1976).</li><li>Kovacs, H., Moskau, D., Spraul, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cryogenically+cooled+probes+-+a+leap+in+NMR+technology.">Cryogenically cooled probes - a leap in NMR technology.</a> Prog. Nucl. Magn. Reson. Spectrosc. 46 (2-3), 131-155 (2005).</li><li>Waiczies, H., Millward, J. 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The authors declare that they have no competing financial interests.

This method of employing 19F/1H MRI to follow the movement of DC into the lymph node gives the opportunity to study the migration patterns of immune cells in vivo. Dendritic cells are excellent examples of rapidly migrating immune cells that are able to maneuver through three-dimensional structures without tightly adhering to specific substrates 17. Although the low spatial resolution (&mu;m range) of the described technique is not comparable with the high resolution (nm ra...

The tracking of cells in vivo is a crucial aspect in several fields of biomedicine. For this, noninvasive imaging techniques that can selectively localize cells over a period of time are extremely valuable. Prior to the development of three-dimensional magnetic resonance imaging (MRI), the tracking of immune cell migration was limited to microscopic analyses or tissue biopsies. Cell tracking with the help of MRI has gained immense attention in the past few years, not only for immunologists studying immune cell behavior in vivo, but also for clinical and stem cell researchers. During the mid-90s, the first studies on iron oxide nanoparticles 1 initiated a cascade of developments for tracking cells with MRI. Iron oxide particles shorten the MR relaxation time (T2*) of the labeled cells and thus cause signal depletion in MR images. Iron oxide particles have been employed to label macrophages 2, oligodendrocyte progenitors 3 and many other cell types. Some of these particles have also been clinically approved by the FDA for labeling cellular vaccines in melanoma patients 4. Since in vivo or ex vivo labeling of cells with iron oxide particles relies on a shortening of the T2* signal and the latter could be also brought about by in vivo susceptibility-related T2* effects such as micro bleeds, iron deposits or air bubbles, it might be difficult to identify labeled cells in vivo from other background T2* signal extinctions 5.
In this article, we describe a technique for tracking dendritic cells (DC) in vivo by employing 19F/1H magnetic resonance imaging (MRI). This cell tracking technology was only introduced in 2005 6, several years after the first recognized applications for 19F in MRI had been reported 7. One important advantage of 19F over iron oxide particle cell labeling is the low biological occurrence of 19F in tissue; this makes it possible to track cells very selectively with basically background-free images. Furthermore, it is possible to overlay the 19F MR signal from the transplanted labeled cells with anatomical images obtained from conventional 1H MRI. 19F/1H MRI is therefore considerably relevant for studies investigating cell migration in vivo. Cells studied with this method are labeled with 19F-rich particles. Synthetically-derived perfluorocarbons (PFCs) consisting primarily of carbon and fluorine atoms are commonly used to prepare the particles. These compounds are insoluble in water and need to be emulsified prior to application in vitro or in vivo. The usual size of the PFC particles that have been employed by other groups for in vivo 19F-MRI tracking experiments ranges between 100 nm and 245 nm 6,8-10. We have however shown that the efficiency in labeling dendritic cells with perfluoro-15-crown-5-ether (PFCE) particles increases with increasing particle size (&gt;560 nm).11

<table border="1">
 <tbody>
 <tr>
 <td></td>
 <td></td>
 <td></td>
 <td>REAGENTS</td>
 </tr>
 <tr>
 <td>C57BL/6 mice</td>
 <td>Charles River, Berlin</td>
 <td></td>
 <td></td>
 </tr>
 <tr>
 <td>RPMI</td>
 <td>Gibco</td>
 <td>21875-091</td>
 <td></td>
 </tr>
 <tr>
 <td>FBS Superior</td>
 <td>Biochrom AG</td>
 <td>S 0615</td>
 <td></td>
 </tr>
 <tr>
 <td>HEPES</td>
 <td>Gibco-Invitrogen</td>
 <td>15630-056</td>
 <td></td>
 </tr>
 <tr>
 <td>Penicillin-Streptomycin</td>
 <td>Gibco</td>
 <td>15140-122</td>
 <td></td>
 </tr>
 <tr>
 <td>L-glutamine</td>
 <td>Gibco</td>
 <td>25030-024</td>
 <td></td>
 </tr>
 <tr>
 <td>Dulbecco's PBS</td>
 <td>Sigma Aldrich</td>
 <td>D8662</td>
 <td></td>
 </tr>
 <tr>
 <td>PFA</td>
 <td>Santa Cruz</td>
 <td>sc-281692</td>
 <td></td>
 </tr>
 <tr>
 <td>Perfluoro-15-crown-5-ether </td>
 <td>ChemPur</td>
 <td>391-1996</td>
 <td></td>
 </tr>
 <tr>
 <td>Pluronic F-68 </td>
 <td>Sigma Aldrich</td>
 <td>P5556</td>
 <td></td>
 </tr>
 <tr>
 <td>Petri dishes (35 x 10 mm)</td>
 <td>VWR, Germany</td>
 <td>391-1996</td>
 <td></td>
 </tr>
 <tr>
 <td>27 &frac12; G syringes</td>
 <td>VWR, Germany</td>
 <td>612-0151</td>
 <td></td>
 </tr>
 <tr>
 <td>Nylon cell strainers (100 &mu;m mesh) </td>
 <td>VWR, Germany</td>
 <td>734-0004</td>
 <td></td>
 </tr>
 <tr>
 <td>NMR tubes </td>
 <td>VWR, Germany</td>
 <td>634-0461</td>
 <td></td>
 </tr>
 <tr>
 <td></td>
 <td></td>
 <td></td>
 <td>EQUIPMENT</td>
 </tr>
 <tr>
 <td>Dissection tools</td>
 <td>FST</td>
 <td></td>
 <td></td>
 </tr>
 <tr>
 <td>CO2 incubator </td>
 <td>Binder</td>
 <td></td>
 <td></td>
 </tr>
 <tr>
 <td>Small animal MR system</td>
 <td>Bruker Biospin</td>
 <td></td>
 <td>9.4T BioSpec 94/20 USR, ParaVision Acquisition and Processing Software</td>
 </tr>
 <tr>
 <td>1H/19F dual-tunable volume RF coil </td>
 <td>Rapid Biomed, W&uuml;rzburg, Germany</td>
 <td></td>
 <td>35 mm inner diameter, 50 mm length</td>
 </tr>
 <tr>
 <td>19F spectroscopy coil </td>
 <td>in-house</td>
 <td></td>
 <td>tune/match loop coil, 4 turns, inner diameter 5 mm, 10 mm long, two capacitors for tuning and matching</td>
 </tr>
 <tr>
 <td>Isoflurane inhalation system</td>
 <td>F&ouml;hr Medical Instruments GmbH</td>
 <td></td>
 <td></td>
 </tr>
 <tr>
 <td>Animal monitoring system Model 1025</td>
 <td>SA Instruments Inc., New York, USA</td>
 <td></td>
 <td></td>
 </tr>
 </tbody>
</table>

monitoring dendritic cell migration using 19f / 1h magnetic resonance imaging

Continuous advancements in noninvasive imaging modalities such as magnetic resonance imaging (MRI) have greatly improved our ability to study physiological or pathological processes in living organisms. MRI is also proving to be a valuable tool for capturing transplanted cells in vivo. Initial cell labeling strategies for MRI made use of contrast agents that influence the MR relaxation times (T1, T2, T2*) and lead to an enhancement (T1) or depletion (T2*) of signal where labeled cells are present. T2* enhancement agents such as ultrasmall iron oxide agents (USPIO) have been employed to study cell migration and some have also been approved by the FDA for clinical application. A drawback of T2* agents is the difficulty to distinguish the signal extinction created by the labeled cells from other artifacts such as blood clots, micro bleeds or air bubbles. In this article, we describe an emerging technique for tracking cells in vivo that is based on labeling the cells with fluorine (19F)-rich particles. These particles are prepared by emulsifying perfluorocarbon (PFC) compounds and then used to label cells, which subsequently can be imaged by 19F MRI. Important advantages of PFCs for cell tracking in vivo include (i) the absence of carbon-bound 19F in vivo, which then yields background-free images and complete cell selectivityand(ii) the possibility to quantify the cell signal by 19F MR spectroscopy.

Eighteen to twenty-one hours following intracutaneous application, 19F-labeled dendritic cells (DC) migrate into the draining popliteal lymph node. The movement of DC via the lymphatic ducts into the draining politeal lymph node can be appreciated by overlaying the 1H anatomical images with the 19F DC images (Figure 2A). We have previously reported on the migration of these cells in vivo, as well as the impact of 19F-particle size on DC immunobiology, ...

Watch this Scientific Journal Video about Monitoring Dendritic Cell Migration using 19F / 1H Magnetic Resonance Imaging at JoVE.com

Monitoring Dendritic Cell Migration using 19F / 1H Magnetic Resonance Imaging

Tracking of cells using MRI has gained remarkable attention in the past years. This protocol describes the labeling of dendritic cells with fluorine (19F)-rich particles, the in vivo application of these cells, and monitoring the extent of their migration to the draining lymph node with 19F/1H MRI and 19F MRS.

Monitoring Dendritic Cell Migration using 19F / 1H Magnetic Resonance Imaging

Continuous  advancements in noninvasive imaging modalities such as magnetic resonance  imaging (MRI) have greatly improved our ability to study ...

monitoring-dendritic-cell-migration-using-19f-1h-magnetic-resonance

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JoVE Journal

Immunology and Infection

11.7K Görüntüleme.  A joint cooperation between the Charité Medical Faculty and the Max Delbrück Center for Molecular Medicine. MRI kullanarak hücrelerin Takip geçmiş yıllarda kayda değer bir ilgi kazanmıştır. Bu protokol, flor ile dendritik hücrelerin etiketlenmesi açıklanmaktadır ( 19 F) zengin bu hücrelerin in vivo uygulama parçacıklar, ve ile drenaj lenf nodu göç ölçüde izlenmesi 19 F / 1 H MR ve 19 F MRS.