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 described by the group of Ralph M. Steinman (1943-2011), discoverer of the dendritic cell 14.</li>
	<li>Briefly, extract tibia and fibula soon after sacrificing mice. Working under the laminar flow hood (to prevent microbial contamination), flush the bone marrow into a small Petri dish with wash medium (5% FBS, 1% penicillin/streptomycin and 1% HEPES in RPMI) 12.</li>
	<li>Transfer the cell suspension through a cell strainer (100 &mu;m mesh size, nylon) into a sterile 50-ml centrifuge tube in order to remove bone and leftover tissue. After several wash steps and a lysis step 12, count the viable cells using trypan blue and a hemocytometer.</li>
	<li>Resuspend bone marrow cells at a concentration of 400,000 cells ml-1 in culture medium (RPMI supplemented with 10% FBS, 1% penicillin/streptomycin, 1% HEPES and 1% L-glutamine) containing 75 ng ml-1 of the mouse granulocyte macrophage-colony stimulating factor (mGM-CSF) obtained from supernatants of 293FT HEK cells transfected with a mouse GMCSF plasmid. Transfer 10 ml of cell suspension (4 x 106 cells) in Petri dishes (100 mm x 20 mm) and incubate in a CO2 incubator (37 &deg;C, 5% CO2). On day 3, replenish old medium with 10 ml fresh medium (keeping end concentration of mGM-CSF at 75 ng ml-1). On day 6, remove 10 ml of old medium and replenish with 10 ml fresh medium (mGM-CSF end concentration = 75 ng ml-1). On day 8, remove 10 ml of old medium and replenish with 10 ml fresh medium (mGM-CSF end concentration = 150 ng ml-1).</li>
	<li>On day 10, mature the differentiated DC with 1 &mu;g ml-1 lipopolysaccharide (LPS) for 24 hr.</li>
</ol>
2. Labeling of Dendritic Cells with 19F-rich Particles
<ol>
	<li>During the 24 hr maturation step (1.5), label the DC with 1 mM fluorine-rich perfluoro-15-crown-5-ether (PFCE) particles (500-560 nm) also for 24 hr. Prepare large fluorine-labeled particles by emulsifying PFCE in Pluronic F-68 (total volume 2.5 ml) using a cell-disrupting titanium sonotrode and employing a continuous pulse program (amplitude 100%, power = 250 W) for 60 sec.</li>
	<li>Following the above incubation steps, harvest the mature and fluorine-labeled DC using a tissue culture cell scraper, transfer into a 50-ml centrifuge tube and centrifuge at 500 &times; g for 5 min at room temperature.</li>
	<li>To wash off any leftover particles and dead cells, leave harvested cells to adhere on poly-L-lysine dishes. To prepare for this step, coat Petri dishes with poly-L-lysine (1 mg ml-1) at 37 &deg;C for 1 hr, thereafter wash off the unbound poly-L-lysine with PBS.</li>
	<li>Incubate above harvested cells on the poly-L-lysine-coated plates in serum-free medium and allow cells to adhere (1 hr at 37 &deg;C, 5% CO2).</li>
	<li>Wash off the medium to discard non-adherent cells, remaining particles and dead cells and wash three times with pre-warmed (37 &deg;C) PBS.</li>
	<li>Harvest adherent cells by trypsin-EDTA treatment (0.25% trypsin-EDTA, 3 min incubation at 37 &deg;C).</li>
	<li>Following another wash centrifugation step, resuspend the required amount of mature DC in serum-free sterile PBS.</li>
	<li>To determine whether the cells have been successfully labeled, it is recommended to check the physicochemical characteristics of the cells using flow cytometry (FACS). The sideward scatter (SSC) should reveal an increased granularity in these cells, as previously described 11. It should be also kept in mind that fibroblasts might contaminate the DC cultures and could also take up the 19F particles in parallel. Therefore the DC purity should be assessed (by measuring the percentage of CD11c+CD11b+ cells using FACS) prior to in vivo application.</li>
</ol>
3. In Vivo Application of 19F-labeled Dendritic Cells
<ol>
	<li>Administer DC (106 - 107) in a volume of 50 - 100 &mu;l intracutaneously into the hind limbs of a C57BL/6 mouse by positioning the mouse in a holder and carefully inserting a 26 &frac12; G needle into the upper layers of the skin prior to application (use 0.5 ml tuberculin syringes with permanently-attached needles to minimize dead volume). To ensure an appropriate intracutaneous application, it is important that the inserted part of the needle be partially visible beneath the skin. If the needle is not visible any more, the needle has been inserted too deep into the skin layers. For the untrained individual, the intracutaneous application could be performed under anesthesia.</li>
	<li>Image the limbs by in vivo fluorine (19F) / proton (1H) MRI (see next section) 21 hr following injection.</li>
</ol>
4. In Vivo 19F/1H Magnetic Resonance Imaging
The detailed instructions for setting up the MR scans refer to a Bruker MR scanner using the control software Paravison (version 5.1). Names referring to vendor-specific functions and items have been highlighted in italics. For other MR scanners these steps may have to be adjusted according to the manufacturers' guidelines.
<ol>
	<li>Arrange access to a dedicated small animal MR scanner. For adequate image quality, a system with a magnetic field strength of 9.4 Tesla or more and tailored radio frequency coils (e.g. 1H/19F dual-tunable volume RF coil, 35 mm inner diameter, 50 mm length; Rapid Biomed, W&uuml;rzburg, Germany) are recommended.</li>
	<li>Prior to MRI, prepare the setup of the MRI scanner for anesthesia and physiological monitoring. Ensure that the breathing mask of the animal bed/holder is connected to the isoflurane system and that the temperature probe and breathing pad are connected to a remote animal monitoring system (e.g. Model 1025, SA Instruments Inc., New York, USA). The latter serves to monitor the animal's vital parameters such as body temperature and respiration.</li>
	<li>Anesthetize mice by inhalation narcosis using a mouse chamber connected to an isoflurane system. Adjust the flow rate for air and O2 at 0.2 L min-1 and 0.1 L min-1, respectively and 3% isoflurane (adjusted from a vaporizer) for about 2 min until the required level of anesthesia is reached (no response following toe pinch). Optimal flow rates may vary depending on the setup being used. Be aware that high flow rates might lead to dehydration. Careful monitoring of the animals' conditions is required at all times as mentioned above.</li>
	<li>Position the mouse prone on the mouse bed/holder of the small animal MR scanner (Figure 1).</li>
	<li>While keeping the flow rate for air and O2 constant, adjust the isoflurane vaporizer to 0.8 - 1.5% until an optimal breathing pattern is reached. A respiratory rate of 50 - 70 breaths per min is recommended.</li>
	<li>Keep the body temperature at 36-37 &deg;C during the experiment by employing a warm water (or alternatively warm air) circulation system.</li>
	<li>Keep the eyes of the mouse moist with sterile eye lubricating ointment during the measurement.</li>
	<li>After securing the animal on the imaging holder and the connections to the monitoring system, move the holder to the center of the 1H/19F RF coil (that is positioned at the isocenter of the animal scanner magnet).</li>
	<li>Set the position of the mouse such that the knee lies within the isocenter of the magnet, either with an automated method (e.g. using a positioning laser combined with a motor-driven animal holder) or by manually calculating the distance the holder needs to be moved into the magnet to reach the isocenter.</li>
	<li>For confirming the correct positioning of the animal in the scanner and later planning of the image slice geometry (i.e. size, position and rotation in space), acquire scout images in three standard orientations (axial, coronal, sagittal) using fast and low resolution image acquisition methods (e.g. TriPilot protocol, which uses a FLASH pulse sequence).</li>
	<li>Tune the RF coil to the 1H resonance frequency (e.g. 400.1 MHz for 9.4 T) and to the 19F resonance frequency (e.g. 376.3 MHz for 9.4 T) and match the characteristic impedance of the coil to 50 Ohm using the tuning monitor of the animal MR scanner. Tuning and matching is necessary to achieve the optimum conditions for transmission and signal reception.</li>
	<li>Perform the necessary automatic system settings including shimming to fine-tune the homogeneity of the magnetic field (e.g. ADJ_SHIM), system frequency adjustments to tune the RF to the Lamor frequency of the mouse (e.g. ADJ_SF) and reference gain to adjust the RF amplitude (e.g. ADJ_REFG). All these settings are important to make the static and variable magnetics fields (B0, B1) in the region of interest as homogeneous as possible.</li>
	<li>Acquire a second set of scout images (as above) along the three orthogonal orientations, after having adjusted the field of view (FOV) such that both lower limbs are visible from pelvis to foot (LxWxH = 50x25x25 mm). These images serve to plan the orientations of the final 3D 1H/19F scans.</li>
	<li>Set up a TurboRARE 3D protocol for 1H-scan: TR/TE = 1,500/53 msec, FOV = 50x25x25 mm, Matrix = 400x200x200, RARE Factor = 16 (scan time approx. 60 min). Adjust FOV with the help of the scout images, start the scan (traffic-light).</li>
	<li>Load a single pulse FID-sequence with a TR of at least 1,000 msec. Open Edit Scan and set nucleus to 19F. Use manual gain settings by deselecting the automatic reference gain in the Edit Method of the Toolbox.</li>
	<li>Start measurement without recording data to display an MR signal in real time by using GSP (go setup). If the 19F spectral signal within the acquisition window is too low, add more averages in Edit Method and/or modulate the pulse attenuator (TX0 or SP0 slider) until a signal is clearly visible. Adjust the basic frequency in order to center the 19F spectral peak at 0 Hz in the acquisition window. Apply basic frequency and press Stop. Note that the isoflurane used as anesthesia may generate a background signal. At 9.4 Tesla MRI the chemical shift between the 19F-containing particles and the isoflurane is about 1,800 Hz. Ensure that the excitation bandwidth is smaller than 3,600 Hz to avoid exciting the isoflurane together with the 19F-labeled cells or particles. The basic frequency should be set correctly on the signal generated by the 19F-labeled cells. Alternatively, another method of anesthesia could be used as the experimenter sees fit.</li>
	<li>Clone (duplicate) the TurboRARE 3D protocol from the previous 1H scan. Go to Edit Method and deselect the automatic reference gain (as above). Set 19F nucleus from Edit Scan. Change the matrix to 128x64x64 and the RARE-Factor to at least 40 (up to 64). For a TR/TE 1,000/6 msec and 64 averages, the scan time is approximately 70 min. Set the receiver gain to 101 (toolbox) and start the scan without any further adjustments using GOP (go pipeline).</li>
	<li>When the scans are finished retract the mouse holder from the MR scanner. Disconnect the mouse carefully from the holder. If the mouse is not sacrificed for ex vivo analysis (e.g. histology or spectroscopy, see below) directly after the MR measurements, closely monitor until it has completely recovered from anesthesia. Body temperature regulation is affected by the anesthesia, so during the recovery process, put the mouse in a separate cage that is placed on a warm temperature-regulated pad. Once the mouse has completely recovered from anesthesia, return it to its holding cage and to the animal room.</li>
	<li>To overlay the 19F images to 1H images, use the View Menu option on the same software (Paravison). The Underlay function can be found under Correlate. Save the underlay, load the new image and highlight the 19F layer by defining a new color from the Look up table. To discriminate between the 19F and 1H scans, alter the threshold for the chosen color (cut Look up table) such that the 19F signal will have the chosen color and the background 1H image will remain in grayscale. Other post-processing programs (e.g. ImageJ) are available to create the 1H/19F image overlays.</li>
	<li>Should an in vivo quantification of the 19F signal be necessary, follow a simple quantitative protocol as previously described 15,16. In brief, quantification can be done by placing a reference tube with a known concentration of PFCE-Emulsion within the FOV next to the mouse. After acquiring the MR images, quantify the intensities of the 19F signals using region-of-interest (ROI) analysis and compare to the in vitro calibration curve as shown in Figure 2.</li>
</ol>
5. Lymph Node Magnetic Resonance Spectroscopy (MRS)
<ol>
	<li>After sacrificing the mouse, remove the popliteal lymph node and place in a small 5 mm NMR tube and add 100 &mu;l of 2% PFA.</li>
	<li>Install a 19F spectroscopy coil (e.g. a toroidal coil) which has an opening of at least 5 mm for holding the NMR tube containing the lymph node.</li>
	<li>Place the NMR tube inside the coil and move the coil into the isocenter of the magnet.</li>
	<li>Tune the RF coil to the 19F resonance frequency (e.g. 376.3 MHz for 9.4 T) and match the characteristic impedance of the coil to 50 Ohm using the tuning monitor of the animal MR scanner. Tuning and matching is necessary to achieve the optimum conditions for transmission and signal reception.</li>
	<li>Set all shim parameters within the Shimming Toolbox to 0 and press apply. Usually it is not necessary to apply shimming methods since the volume of the sample is relatively small. If a sufficient 19F signal is available, automated methods may be applied for shimming, system frequency and receiver gain as described above.</li>
	<li>Load a single pulse FID sequence with a TR of at least 1,000 msec. Open Edit Scan and set the nucleus to 19F. Use manual gain settings by deselecting the automatic reference gain in the Edit Method of the Toolbox. Set the number of averages to 1.</li>
	<li>Start the sequence using GSP. If the 19F spectral signal within the acquisition window is too low, add more averages in Edit Method and/or modulate the pulse attenuator (TX0 or SP0 slider) until a signal is clearly visible. Adjust the basic frequency so that the 19F spectral peak is at the center of the acquisition window at 0 Hz and apply basic frequency. Adjust the pulse attenuator again to maximize the signal, to reach 90 &deg; excitation. When signal is at maximum press Stop.</li>
	<li>Set the averages in Edit Method between 64 and 256 (or more if necessary, depending on the signal) and start the acquisition using GOP. The detected signal correlates linearly with the number of averages. During quantification keep in mind that the detected signal needs to be normalized to one average. Furthermore, a calibration standard of a known concentration or a calibration curve is necessary to calculate the number of cells from the normalized 19F signal (Figure 2).</li>
	<li>Once the scan is finished, remove the sample, place the next sample and proceed with step 5.3.</li>
</ol>

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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">
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 <td>REAGENTS</td>
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 <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

Research

JoVE Journal

Immunology and Infection

11.7K Views.  A joint cooperation between the Charité Medical Faculty and the Max Delbrück Center for Molecular Medicine. 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.

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

Non-invasive Imaging of Leukocyte Homing and Migration in vivo

Two-photon (2P) microscopy is a high resolution imaging technique that has been broadly adapted by biologists. The value of 2P microscopy is that it provides rich spatiotemporal information regarding cell behaviors within intact tissues and in live mice. Leukocyte recruitment plays a significant role in host defense against infection and when unchecked, can contribute to inflammatory and autoimmune diseases. Studying leukocyte recruitment in vivo is technically challenging since cells are moving rapidly within vessels located deep within light scattering tissues. To date, most intravital imaging studies require surgical preparation to expose the blood vessels and tissues. To avoid the tissue damage and inflammation induced by surgery itself, here, we describe a non-invasive single-cell imaging approach that can be used to study leukocyte trafficking in the mouse footpad and phalanges. We discuss the technical aspects of our 2P imaging preparation and walk the reader through a typical experiment from initial set up to execution and data collection.

Non-invasive Imaging of Leukocyte Homing and Migration in vivo

Here, we describe a non-invasive two-photon (2P) microscopy approach to study leukocyte homing in the mouse footpad. We discuss the technical aspects of our tissue imaging preparation and walk the reader through a typical experiment from initial set up to execution and data collection.

Two-photon (2P) microscopy is a high resolution imaging technique that has been broadly adapted by biologists.  The value of 2P microscopy is that it ...

Real-time Imaging of Leukotriene B4 Mediated Cell Migration and BLT1 Interactions with &beta;-arrestin

G-protein coupled receptors (GPCRs) belong to the seven transmembrane protein family and mediate the transduction of extracellular signals to intracellular responses. GPCRs control diverse biological functions such as chemotaxis, intracellular calcium release, gene regulation in a ligand dependent manner via heterotrimeric G-proteins1-2. Ligand binding induces a series of conformational changes leading to activation of heterotrimeric G-proteins that modulate levels of second messengers such as cyclic adenosine monophosphate (cAMP), inositol triphosphate (IP3) and diacyl glycerol (DG). Concomitant with activation of the receptor ligand binding also initiates a series of events to attenuate the receptor signaling via desensitization, sequestration and/or internalization. The desensitization process of GPCRs occurs via receptor phosphorylation by G-protein receptor kinases (GRKs) and subsequent binding of &beta;-arrestins3. &beta;-arrestins are cytosolic proteins and translocate to membrane upon GPCR activation, binding to phosphorylated receptors (most cases) there by facilitating receptor internalization 4-6.
Leukotriene B4 (LTB4) is a pro-inflammatory lipid molecule derived from arachidonic acid pathway and mediates its actions via GPCRs, LTB4 receptor 1 (BLT1; a high affinity receptor) and LTB4 receptor 2 (BLT2; a low affinity receptor)7-9. The LTB4-BLT1 pathway has been shown to be critical in several inflammatory diseases including, asthma, arthritis and atherosclerosis10-17. The current paper describes the methodologies developed to monitor LTB4-induced leukocyte migration and the interactions of BLT1 with &beta;-arrestin and , receptor translocation in live cells using microscopy imaging techniques18-19.
Bone marrow derived dendritic cells from C57BL/6 mice were isolated and cultured as previously described 20-21. These cells were tested in live cell imaging methods to demonstrate LTB4 induced cell migration. The human BLT1 was tagged with red fluorescent protein (BLT1-RFP) at C-terminus and &beta;-arrestin1 tagged with green fluorescent protein (&beta;-arr-GFP) and transfected the both plasmids into Rat Basophilic Leukomia (RBL-2H3) cell lines18-19. The kinetics of interaction between these proteins and localization were monitored using live cell video microscopy. The methodologies in the current paper describe the use of microscopic techniques to investigate the functional responses of G-protein coupled receptors in live cells. The current paper also describes the use of Metamorph software to quantify the fluorescence intensities to determine the kinetics of receptor and cytosolic protein interactions.

Real-time Imaging of Leukotriene B4 Mediated Cell Migration and BLT1 Interactions with &amp;#946;-arrestin

This paper describes the methodology to determine the chemotactic response of leukocytes to specific ligands and identify interactions between the cell surface receptors and cytosolic proteins using live cell imaging techniques.

G-protein coupled receptors (GPCRs) belong to the seven transmembrane protein family and mediate the transduction of extracellular signals to ...

Competitive Homing Assays to Study Gut-tropic T Cell Migration

In order to exert their function lymphocytes need to leave the blood and migrate into different tissues in the body. Lymphocyte adhesion to endothelial cells and tissue extravasation is a multistep process controlled by different adhesion molecules (homing receptors) expressed on lymphocytes and their respective ligands (addressins) displayed on endothelial cells 1 2. Even though the function of these adhesion receptors can be partially studied ex vivo, the ultimate test for their physiological relevance is to assess their role during in vivo lymphocyte adhesion and migration. Two complementary strategies have been used for this purpose: intravital microscopy (IVM) and homing experiments. Although IVM has been essential to define the precise contribution of specific adhesion receptors during the adhesion cascade in real time and in different tissues, IVM is time consuming and labor intensive, it often requires the development of sophisticated surgical techniques, it needs prior isolation of homogeneous cell populations and it permits the analysis of only one tissue/organ at any given time. By contrast, competitive homing experiments allow the direct and simultaneous comparison in the migration of two (or even more) cell subsets in the same mouse and they also permit the analysis of many tissues and of a high number of cells in the same experiment.
Here we describe the classical competitive homing protocol used to determine the advantage/disadvantage of a given cell type to home to specific tissues as compared to a control cell population. We chose to illustrate the migratory properties of gut-tropic versus non gut-tropic T cells, because the intestinal mucosa is the largest body surface in contact with the external environment and it is also the extra-lymphoid tissue with the best-defined migratory requirements. Moreover, recent work has determined that the vitamin A metabolite all-trans retinoic acid (RA) is the main molecular mechanism responsible for inducing gut-specific adhesion receptors (integrin a4b7and chemokine receptor CCR9) on lymphocytes. Thus, we can readily generate large numbers of gut-tropic and non gut-tropic lymphocytes ex vivoby activating T cells in the presence or absence of RA, respectively, which can be finally used in the competitive homing experiments described here.

Competitive homing experiments allow to directly assessing the migratory properties of two different cell populations in a single mouse. Here we illustrate this procedure by comparing the migration of ex vivo-generated gut-tropic versus non-gut tropic T cells.

In order to exert their function lymphocytes need to leave the blood and migrate into different tissues in the body. Lymphocyte adhesion to endothelial ...

Optimized Protocol for Efficient Transfection of Dendritic Cells without Cell Maturation

Dendritic cells (DCs) can be considered sentinels of the immune system which play a critical role in its initiation and response to infection1. Detection of pathogenic antigen by na&iuml;ve DCs is through pattern recognition receptors (PRRs) which are able to recognize specific conserved structures referred to as pathogen-associated molecular patterns (PAMPS). Detection of PAMPs by DCs triggers an intracellular signaling cascade resulting in their activation and transformation to mature DCs. This process is typically characterized by production of type 1 interferon along with other proinflammatory cytokines, upregulation of cell surface markers such as MHCII and CD86 and migration of the mature DC to draining lymph nodes, where interaction with T cells initiates the adaptive immune response2,3. Thus, DCs link the innate and adaptive immune systems. 
The ability to dissect the molecular networks underlying DC response to various pathogens is crucial to a better understanding of the regulation of these signaling pathways and their induced genes. It should also help facilitate the development of DC-based vaccines against infectious diseases and tumors. However, this line of research has been severely impeded by the difficulty of transfecting primary DCs4.
Virus transduction methods, such as the lentiviral system, are typically used, but carry many limitations such as complexity and bio-hazardous risk (with the associated costs)5,6,7,8. Additionally, the delivery of viral gene products increases the immunogenicity of those transduced DCs9,10,11,12. Electroporation has been used with mixed results13,14,15, but we are the first to report the use of a high-throughput transfection protocol and conclusively demonstrate its utility.
In this report we summarize an optimized commercial protocol for high-throughput transfection of human primary DCs, with limited cell toxicity and an absence of DC maturation16. Transfection efficiency (of GFP plasmid) and cell viability were more than 50% and 70% respectively. FACS analysis established the absence of increase in expression of the maturation markers CD86 and MHCII in transfected cells, while qRT-PCR demonstrated no upregulation of IFN&beta;. Using this electroporation protocol, we provide evidence for successful transfection of DCs with siRNA and effective knock down of targeted gene RIG-I, a key viral recognition receptor16,17, at both the mRNA and protein levels.

We present our optimized high-throughput nucleofection protocol as an efficient way of transfecting primary human monocyte-derived dendritic cells with either plasmid DNA or siRNA without causing cell maturation. We further provide evidence for successful siRNA silencing of targeted gene RIG-I at both the mRNA and protein levels.

Dendritic cells (DCs) can be considered sentinels of the immune system which play a critical role in its initiation and response to infection1. Detection ...

Sample Preparation of Mycobacterium tuberculosis Extracts for Nuclear Magnetic Resonance Metabolomic Studies

Mycobacterium tuberculosis is a major cause of mortality in human beings on a global scale. The emergence of both multi- (MDR) and extensively-(XDR) drug-resistant strains threatens to derail current disease control efforts. Thus, there is an urgent need to develop drugs and vaccines that are more effective than those currently available. The genome of M. tuberculosis has been known for more than 10 years, yet there are important gaps in our knowledge of gene function and essentiality. Many studies have since used gene expression analysis at both the transcriptomic and proteomic levels to determine the effects of drugs, oxidants, and growth conditions on the global patterns of gene expression. Ultimately, the final response of these changes is reflected in the metabolic composition of the bacterium including a few thousand small molecular weight chemicals. Comparing the metabolic profiles of wild type and mutant strains, either untreated or treated with a particular drug, can effectively allow target identification and may lead to the development of novel inhibitors with anti-tubercular activity. Likewise, the effects of two or more conditions on the metabolome can also be assessed. Nuclear magnetic resonance (NMR) is a powerful technology that is used to identify and quantify metabolic intermediates. In this protocol, procedures for the preparation of M. tuberculosis cell extracts for NMR metabolomic analysis are described. Cell cultures are grown under appropriate conditions and required Biosafety Level 3 containment,1 harvested, and subjected to mechanical lysis while maintaining cold temperatures to maximize preservation of metabolites. Cell lysates are recovered, filtered sterilized, and stored at ultra-low temperatures. Aliquots from these cell extracts are plated on Middlebrook 7H9 agar for colony-forming units to verify absence of viable cells. Upon two months of incubation at 37 &deg;C, if no viable colonies are observed, samples are removed from the containment facility for downstream processing. Extracts are lyophilized, resuspended in deuterated buffer and injected in the NMR instrument, capturing spectroscopic data that is then subjected to statistical analysis. The procedures described can be applied for both one-dimensional (1D) 1H NMR and two-dimensional (2D) 1H-13C NMR analyses. This methodology provides more reliable small molecular weight metabolite identification and more reliable and sensitive quantitative analyses of cell extract metabolic compositions than chromatographic methods. Variations of the procedure described following the cell lysis step can also be adapted for parallel proteomic analysis.

Sample Preparation of Mycobacterium tuberculosis Extracts for Nuclear Magnetic Resonance Metabolomic Studies

The metabolomic profile of Mycobacterium tuberculosis is determined after growth in broth cultures. Conditions can be varied to test the effects of nutritional supplements, oxidants, and anti-tuberculosis agents on the metabolic profile of this microorganism. Procedure for extract preparation is applicable for both 1D 1H and 2D 1H-13C NMR analyses.

Mycobacterium tuberculosis is a major cause of mortality in human beings on a global scale. The emergence of both multi- (MDR) and extensively-(XDR) ...

Analysis of Pulmonary Dendritic Cell Maturation and Migration during Allergic Airway Inflammation

Dendritic cells (DCs) are the key players involved in initiation of adaptive immune response by activating antigen-specific T cells. DCs are present in peripheral tissues in steady state; however in response to antigen stimulation, DCs take up the antigen and rapidly migrate to the draining lymph nodes where they initiate T cell response against the antigen1,2. Additionally, DCs also play a key role in initiating autoimmune as well as allergic immune response3.
DCs play an essential role in both initiation of immune response and induction of tolerance in the setting of lung environment4. Lung environment is largely tolerogenic, owing to the exposure to vast array of environmental antigens5. However, in some individuals there is a break in tolerance, which leads to induction of allergy and asthma. In this study, we describe a strategy, which can be used to monitor airway DC maturation and migration in response to the antigen used for sensitization. The measurement of airway DC maturation and migration allows for assessment of the kinetics of immune response during airway allergic inflammation and also assists in understanding the magnitude of the subsequent immune response along with the underlying mechanisms.
Our strategy is based on the use of ovalbumin as a sensitizing agent. Ovalbumin-induced allergic asthma is a widely used model to reproduce the airway eosinophilia, pulmonary inflammation and elevated IgE levels found during asthma6,7. After sensitization, mice are challenged by intranasal delivery of FITC labeled ovalbumin, which allows for specific labeling of airway DCs which uptake ovalbumin. Next, using several DC specific markers, we can assess the maturation of these DCs and can also assess their migration to the draining lymph nodes by employing flow cytometry.

We describe a strategy to monitor maturation and migration of pulmonary dendritic cells in response to ovalbumin in the setting of ovalbumin induced allergic airway inflammation. This strategy can be modified to assess migration of pulmonary dendritic cells in settings of infection.

Dendritic cells (DCs) are the key players involved in initiation of adaptive immune response by activating antigen-specific T cells. DCs are present in ...

Generation of a Novel Dendritic-cell Vaccine Using Melanoma and Squamous Cancer Stem Cells

We identified cancer stem cell (CSC)-enriched populations from murine melanoma D5 syngeneic to C57BL/6 mice and the squamous cancer SCC7 syngeneic to C3H mice using ALDEFLUOR/ALDH as a marker, and tested their immunogenicity using the cell lysate as a source of antigens to pulse dendritic cells (DCs). DCs pulsed with ALDHhigh CSC lysates induced significantly higher protective antitumor immunity than DCs pulsed with the lysates of unsorted whole tumor cell lysates in both models and in a lung metastasis setting and a s.c.&#160;tumor growth setting, respectively. This phenomenon was due to CSC vaccine-induced humoral as well as cellular anti-CSC responses. In particular, splenocytes isolated from the host subjected to CSC-DC vaccine produced significantly higher amount of IFN&#947; and GM-CSF than splenocytes isolated from the host subjected to unsorted tumor cell lysate pulsed-DC vaccine. These results support the efforts to develop an autologous CSC-based therapeutic vaccine for clinical use in an adjuvant setting.

Evaluated in syngeneic immunocompetent hosts, cancer stem cell (CSC) based dendritic cell (DC) vaccine demonstrated significantly higher antitumor immunity than traditional DC vaccines pulsed with heterogeneous bulk tumor cells.

We identified cancer stem cell (CSC)-enriched populations from murine melanoma D5 syngeneic to C57BL/6 mice and the squamous cancer SCC7 syngeneic to C3H ...

Imaging CD4 T Cell Interstitial Migration in the Inflamed Dermis

The ability of CD4 T cells to carry out effector functions is dependent upon the rapid and efficient migration of these cells in inflamed peripheral tissues through an as-yet undefined mechanism. The application of multiphoton microscopy to the study of the immune system provides a tool to measure the dynamics of immune responses within intact tissues. Here we present a protocol for non-invasive intravital multiphoton imaging of CD4 T cells in the inflamed mouse ear dermis. Use of a custom imaging platform and a venous catheter allows for the visualization of CD4 T cell dynamics in the dermal interstitium, with the ability to interrogate these cells in real-time via the addition of blocking antibodies to key molecular components involved in motility. This system provides advantages over both in vitro models and surgically invasive imaging procedures. Understanding the pathways used by CD4 T cells for motility may ultimately provide insight into the basic function of CD4 T cells as well as the pathogenesis of both autoimmune diseases and pathology from chronic infections.

The mechanisms that govern the interstitial motility of CD4 effector T cells at sites of inflammation are relatively unknown. We present a non-invasive approach to visualize and manipulate in vitro-primed CD4 T cells in the inflamed ear dermis, allowing for study of the dynamic behavior of these cells in situ.

The ability of CD4 T cells to carry out effector functions is dependent upon the rapid and efficient migration of these cells in inflamed peripheral ...

A CFSE-based Assay to Study the Migration of Murine Skin Dendritic Cells into Draining Lymph Nodes During Infection with Mycobacterium bovis Bacille Calmette-Gu&#233;rin

Dendritic cells (DCs) are important for initiating immune responses, in part through their ability to acquire and shuttle antigen to the draining lymph node (DLN). The mobilization of DCs to the DLN is complex and remains to be fully elucidated during infection. Herein described is the use of an innovative, simple assay that relies on the fluorochrome 5- and 6-carboxyfluorescein diacetate succinimidyl ester (CFSE) to track the migration of DCs during footpad infection with Mycobacterium bovis Bacille Calmette-Gu&#233;rin (BCG) in C57BL/6 mice. This assay enables the characterization of skin DC sub-populations that actively relocate to the draining, popliteal LN in response to BCG. This protocol originates from a BCG model where migratory skin DCs were identified by flow cytometry. The assay is amiable to the study and identification of DCs or other cells that home to the popliteal LN after inoculation of microbes, their metabolites or other inflammatory stimuli in the footpad, and consequently to study factors that regulate the migration of these cells.

A CFSE-based Assay to Study the Migration of Murine Skin Dendritic Cells into Draining Lymph Nodes During Infection with &lt;em&gt;Mycobacterium bovis&lt;/em&gt; Bacille Calmette-Gu&amp;#233;rin

An assay in mice to track cell migration from skin to draining lymph node is described which enables the characterization of skin Dendritic cells mobilized to the lymph node after footpad infection with Bacille Calmette-Gu&#233;rin.

Dendritic cells (DCs) are important for initiating immune responses, in part through their ability to acquire and shuttle antigen to the draining lymph ...

Generation of Large Numbers of Myeloid Progenitors and Dendritic Cell Precursors from Murine Bone Marrow Using a Novel Cell Sorting Strategy

Cultures of monocyte-derived dendritic cells (moDC) generated from mouse bone marrow using Granulocyte-Macrophage Colony Stimulating Factor (GM-CSF) have recently been recognized to be more heterogeneous than previously appreciated. These cultures routinely contain moDC as well monocyte-derived macrophages (moMac), and even some less developed cells such as monocytes. The goal of this protocol is to provide a consistent method for identification and separation of the many cell types present in these cultures as they develop, so that their specific functions may be further investigated. The sorting strategy presented here separates cells first into four populations based on expression of Ly6C and CD115, both of which are expressed transiently by cells as they develop in GM-CSF-driven culture. These four populations include Common myeloid progenitors or CMP (Ly6C-, CD115-), granulocyte/macrophage progenitors or GMP (Ly6C+, CD115-), monocytes (Ly6C+, CD115+), and monocyte-derived macrophages or moMac (Ly6C-, CD115+). CD11c is also added to the sorting strategy to distinguish two populations within the Ly6C-, CD115- population: CMP (CD11c-) and moDC (CD11c+). Finally, two populations may be further distinguished within the Ly6C-, CD115+ population based on the level of MHC class II expression. MoMacs express lower levels of MHC class II, while a monocyte-derived DC precursor (moDP) expresses higher MHC class II. This method allows for the reliable isolation of several developmentally distinct populations in numbers sufficient for a variety of functional and developmental analyses. We highlight one such functional readout, the differential responses of these cell types to stimulation with Pathogen-Associated Molecular Patterns (PAMPs).

Here we provide a method for identifying and isolating large numbers of GM-CSF driven myeloid cells using high speed cell sorting. Five distinct populations (Common myeloid progenitors, granulocyte/macrophage progenitors, monocytes, monocyte-derived macrophages, and monocyte-derived DCs) can be identified based on Ly6C and CD115 expression.

Cultures of monocyte-derived dendritic cells (moDC) generated from mouse bone marrow using Granulocyte-Macrophage Colony Stimulating Factor (GM-CSF) have ...