The overall goal of the following experiment is to visualize the migration of dendritic cells into the draining lymph node using non-invasive fluorine magnetic resonance imaging or MRI in combination with conventional proton MRI. This is achieved by first preparing murine bone marrow derived dendritic cells and labeling the fully differentiated cells with fluorine rich particles. During the second step, the fluorine labeled cells are administered intra cutaneous into the hind limbs of a mouse, ensuring the efficient transmigration of the administered cells.
Next, the anatomical region of interest is imaged by fluorine, proton MRI, to monitor the migration of the dendritic cells from the skin to the draining lymph nodes. Ultimately, dendritic cell migration from the area of application into the draining lymph node can be visualized by fluorine MRI. The main advantage of this method over other cell tracking techniques such as those requiring iron oxide nanoparticles, is that carbon bound flooring is virtually absent in all living organisms.
This yields complete background free images and a complete cell selectivity following application of fluorine labeled cells in vivo. This method can help answer questions in the field of immunology, such as what is the influence of molecular candidates in the migration of dendritic cells in vivo. Demonstrating the method is Yulia Kovski technician and Helma VIIs, a senior scientist from my laboratory On day 10 of bone marrow dendritic cell culture.
Activate the dendritic cells with LPS for 24 hours. Label the dendritic cells with one millimolar fluorine rich per fluoro 15 crown five ether particles during this time as well, to confirm that the dendritic cells have been successfully labeled, check the physical chemical characteristics of the cells using flow cytometry. The fluorine labeled cells are shown here as red events on the forward and sideward scatter plot and the unlabeled cells are shown as blue events.
Next position, a C 57 black six mouse in a holder and then load a 0.5 milliliter tuberculin syringe with a 26 and a half gauge needle with fluorescently labeled dendritic cells in a 50 to 100 microliter volume. Now carefully insert the needle into the upper layers of the skin in each of the animal's hind limbs to incu administer the cells. After anesthetizing the mouse position the animal prone on the mouse bed of the small animal MR Scanner.
After securing the animal on the imaging holder and the connections to the monitoring system, move the holder to the center of the proton fluorine RF coil for confirming the correct positioning of the animal and the scanner. And later planning of the image slice geometry. Acquire scout images in three standard orientations using fast and low resolution image acquisition methods in the MR Scanner software.
Tune the RF coil to the proton resonance frequency and to the fluorine resonance frequency. Use the tuning monitor of the animal MR Scanner to match the characteristic impedance of the coil to 50 ohms. Next, set the necessary automatic systems settings, including shimming to fine tune the homogeneity of the magnetic field system frequency adjustments to tune the RF to the specific LA frequency of the MR system for protons and reference gain.
To adjust the RF amplitude, set up a turbo rare 3D protocol for the proton scan, using a TR of 1500 milliseconds and TE of 53 milliseconds, a matrix of 400 by 200 by 200 and a rare factor of 16. Adjust the field of view with the help of the scout images. Then start the scan.
Load a single pulse FID sequence with the TR of at least 1000 milliseconds, and then open the edit scan and set the nucleus to fluorine in the edit method of the toolbox. Deselect the automatic reference gain using the go setup. Start the measurement without recording the data to display an MR signal in real time.
Adjust the basic frequency to center the fluorine spectral peak at zero hertz in the acquisition window. Then select, apply basic frequency and press stop now. Clone the turbo rare 3D protocol from the previous proton scan.
Go to edit method and then deselect the automatic reference gain as just shown in edit scan. Set the fluorine nucleus and then change the matrix to 1 28 by 64 by 64 and the rare factor to at least 40. When the scans are finished, retract the mouse holder from the MR scanner and use the view menu to overlay the fluorine images to proton images.
After sacrificing the mouse, remove the popal lymph node of interest and place it into a five millimeter NMR tube containing 100 microliters of 2%PFA. Next, place the tube inside a fluorine spectroscopy coil and move the coil into the ISO center of the magnet. Tune the RF coil to the fluorine resonance frequency, and then on the tuning monitor of the animal MR Scanner, match the characteristic impedance of the coil to 50 ohms within the shimming toolbox.
Set all the shim parameters to zero and press apply. Then load a single pulse FID sequence with a TR of at least 1000 milliseconds. Open edit scan and set the nucleus to fluorine.
Now using go setup, start the sequence, adjust the basic frequency so that the fluorine spectral peak is at the center of the acquisition window at zero hertz and apply the basic frequency. Then adjust the pulse attenuator again to maximize the signal to reach 90 degrees excitation. When the signal is at the maximum, press stop.
Finally, in edit method, set the averages between 64 and 256 depending on the signal, and then using go pipeline. Start the acquisition 18 to 21 hours following intra cutaneous application. Fluorine labeled dendritic cells migrate into the draining popal lymph node.
The movement of the dendritic cells via the lymphatic ducts into the draining publical lymph node can be appreciated by overlaying the proton anatomical images with the fluorine dendritic cells. Images, the dendritic cells are shown as a fluorine MR.Signal in red, whereas the lymph nodes and lymphatic ducts are shown in the proton anatomical MR image in gray scale. In order to quantify the extent of dendritic cell migration into the draining lymph nodes, the lymph nodes are extracted and fluorine MRS is performed as just demonstrated when the fluorine signal obtained from each lymph node is compared with the fluorine signal obtained from different numbers of dendritic cells labeled with per fluoro 15 crown five ether particles as illustrated by this calibration curve, the number of fluorine labeled dendritic cells that reach the draining lymph node can be deduced for this representative experiment.
It can be estimated that 7.5 times 10 to the fourth dendritic cells that were not loaded with antigen reached the left lymph node while 3.6 times 10 to the fifth antigen loaded dendritic cells reach the right lymph node. Though this matters can provide insight into the molecular mechanisms of immune cell migration during normal conditions, it can also be used in animal models such as those of autoimmune diseases. Furthermore, the implications of this technique extend towards the field of cancer therapy.
Any identified molecular targets can be used to manipulate dendritic cells for tumor therapy.
