Tracking Mouse Bone Marrow Monocytes In Vivo

The overall goal of this procedure is to visualize monocyte trafficking in the skull bone marrow of a living mouse using two photon imaging. First, the mouse is anesthetized and injected with a solution of rot, rumine dextran to label the vasculature. The mouse is then placed in a custom metal support, and the scalp from the back of the parietal bone to the frontal bone is removed.

A rubber ring is then applied directly to the skull with surgical glue and the reservoirs filled with liquid saline. Finally, multi photon microscopy is used to track fluorescent cell development over time within the skull bone marrow. This me can help to answer a key question in the field of immunohematology, such as the mechanism of monocyte mobilization in steady state or after chemotherapy.

So this method can provide insight into the behavior of modular monocytes. It can also be applied to study other bone marrow cell subsets depending on the specificity of the flu cent reporter used. The following protocol utilizes a multi photon microscope coupled with a titanium sapphire crystal laser, and an op modulator for laser power control.

To enable simultaneous recording of three fluorescent channels, the configuration should include three external non DS scan detectors or nds with a combination of 565 nanometers and 690 nanometers dichroic mirrors 565, 610 nanometers, and 500 550 nanometer band pass filters, and a 485 short pass filter. Note that in this setup, second harmonic generation or SHG and cyan fluorescent protein or CFP are detected by the NDD after the 485 short pass filter. CFP is also detected by the NDD.

After the 500 550 band pass filter and Rod Domine Dextran is detected by the NDD. After the 565 610 band pass filter, several hours before imaging set the heating chamber to 32 degrees Celsius. This temperature will maintain the mouse at an optimal body temperature throughout the experiment.

Note that the heating chamber used here is dark and covers the objectives and motorized plate of the system. This helps to avoid external light contamination. To turn on the system first, switch on the titanium sapphire laser, then switch on the rest of the microscope system.

Launch the acquisition software. Here, Zeiss Zen software is used. Set the laser wavelength range to 870 nanometers.

Then select the appropriate NDD for recording. The mouse used in this experiment is a transgenic mouse called Mac blue, which expresses cyan fluorescent protein under the control of the CSF one receptor promoter. The recommended anesthesia method depends on the length of imaging, so it is best to consult the accompanying document for details.

A difficult aspect of this procedure is the T vein injection, since it is rather easy to miss the vein. If this happens, perform retroorbital injection instead. Once the mouse is anesthetized, use nippers to check for unconsciousness by stimulating the footpad.Next.

To stain the vasculature inject 200 microliters of rod domine dextran into the tail vein, or for neutrophil staining. Inject 100 microliters of saline containing LY six GPE. After the injection, place the head of the mouse between the retaining plates of a custom made stereotactic holder adapted to the microscope and to the anesthetic gas inhaler.

This ensures that breathing movements will not affect imaging. Tighten the retaining plates to stretch the skin between the ears and the head. Check for stability.

By gently pressing the head with nippers, the head should not move. Then ensure that the animal is breathing normally. To prepare the mouse for two photon imaging, use a finger to apply 70%ethanol to the hair of the scalp, taking care to avoid the eyes.

Then place a drape with a cutout to expose the scalp. Next, using sterile scissors and nippers, cut the skin and fully remove the scalp from the back of the parietal bone to the frontal bone. Maintain a distance of three millimeters from eyes and ears to prevent extensive bleeding.

Remove the loose connective tissue or periosteum covering the skull. Then using a warm PBS soaked paper towel, wash out the remaining hair as shown here. Using a small gauge needle, apply surgical glue to the entire periphery of a 10 millimeter diameter rubber ring.

Then place the rubber ring directly on the skull with another PBS soaked paper towel. Clean the inside the ring one last time. Add warm PBS to completely immerse the skull.

Then place the stereotactic holder under the objective. Once the mouse is in place, use the microscope ocular with direct transmission to view the skull and roughly position it in the microscope field of view. In the upper left of the imaging software window, click on live acquisition.

A live scan of the tissue will appear in the middle of the window using the microscope directional control in the software. Locate a region with CFP and rod domine signal to adjust the focus. Then set the laser power to the minimum to obtain the best signal to noise ratio with minimal photo damage.

Next in the software, click on the channel tab and increase the gain power on the PMTs for each NDD to reduce photo bleaching and photo damage, then click on the appropriate tab to set the acquisition settings to single scanning with a pixel dwell time of 1.58 microseconds and a resolution of 512 by 512 pixels with a 1.5 magnification. Once the recording parameters are set, click on the live acquisition command and survey the tissue to choose a desired x, y, Z field to be monitored in real time. Here, a field including both vascular and parenchymal areas is chosen.

Next to save or register the X, Y, Z coordinates of the field shown in the positions tab. Click on add. Repeat this process with up to three additional fields to register all the coordinates of interest.

The coordinates will appear on the positions tab next to instruct the software. To capture a three DZ stack of images, click on move to in the positions tab in the software to go to the first registered position and select Zack in the upper left box of the software. This will open the Z stack tab, move to the highest Z position, and adjust the laser power accordingly.

Click on set last, then move to the deepest position. Adjust the laser power. Then click on set first.

The thickness of the stack analyze should be adapted to the cell studied fast. Moving cells will go outta French quickly, so you will need to acquire a thicker stack. Then you acquire for slow moving cells.

In the upper left box, select the time series command to open the time series tab. Set the number of cycles and the time interval. Here, one volume will be acquired every 30 seconds for 60 cycles.

This represents 30 minutes in real time. To begin time lapse imaging in the main window, select start experiment throughout the procedure, continue to monitor the animal to make sure it is unconscious. Also ensure the objective is properly immersed.

After the sequence has been captured, use a pipette to add warm PBS. Then begin a second 30 minute acquisition. If progressive disappearance of signal is seen during acquisition, check for PBS leakage.

Once all of the required videos are recorded, performed data analysis as described in the accompanying document to study the bone marrow physiology by intra vital imaging, a Mac blue transgenic mouse was injected with 2 million Dalton rod domine dextran, and imaged by two photon laser scanning microscopy. As demonstrated in this video, in the resulting micrograph, the vasculature stained with 2 million Dalton rod domine dextran is shown in red bone, shown in blue is visualized by second harmonic generation. ECFP positive monocytes shown in cyan are located within parenchymal niches, which are seen as dark areas between vasculature and bone, and within the vessels.

To calculate individual cell trajectory, time-lapse imaging was performed. This video shows the migratory behavior of parenchymal monocytes. ECFP signal is in cyan and migratory paths for each red spot appear in green.

Here, the migratory behavior of vascular monocytes can be seen. These data can be used to calculate both the mean velocity and the mean square displacement of the cells. Note that the vascular monocytes display both increased displacement and increased velocity compared to parenchymal monocytes.

After watching this video, you should have a good understanding of how to track mouse bone marrow monocytes in vivo using two photo imaging After its development. This technique paved the way for researchers in the field of immunology to explore cell migration and interaction in the bone marrow, which represent a very important site of hematopoiesis, but also other fundamental functions such as antigen priming and immunological memory.