The overall goal Of this protocol is to use Brightfield, intravital microscopic and time-lapse video photography to study dynamic neutrophil endothelial cell interactions during neutrophil recruitment in response to neutrophil chemo attractant in vivo. In this video, we demonstrate the procedures of how to use brightfield intravital video microscopic to visualize and determine neutrophil intraluminal crawling, T endothelial migration, and chemotaxis in chma muscle tissue in the mouse. Leukocyte recruitment is the hallmark of inflammation.
This recruitment process initiates when leukocytes in the bloodstream start to roll on the endothelium of post capillary vees at the inflammation site. In response to chemokines or other chemo attractants on the ular surface, these rolling leukocytes arrest on and firmly adhere to the endothelium. After adhesion, many leukocytes crawl on the luminal surface and appear to search for an optimal transmigration site.
Following crawling leukocytes move across the endothelium, a process termed T endothelial migration. Thereafter, neutrophils migrate in the extravascular tissue toward the source of the chemo attractant in the inflammatory site, a process called chemotaxis MOIs. This video shows the experiment of how to use brightfield intra vital video, microscopic time-lapse photography and image J to visualize track and determine neutrophil intraluminal crawling, T endothelial migration and migration in chemotaxis in Oma muscle tissue, and response to the source of a neutrophil chemo Attractant.
Hello, I'm Lis Liu from the Department of Pharmacology College of Medicine at the University of Saskatchewan. Today we're going to show you how to make the neutrophil chemo trant in agro gel. How to prepare mouse re master muscle for intra vital microscopy.
How to track cell movement using MEGJ. And finally, how to analyze the neutrophil recruitment parameters two graduate students in our laboratory, ngel Naja, sh, and Chile will show you the detailed experimental procedures. Hi, I'm Naja Sh.Hi, I am Chile.
To begin this experiment, prepare neutrophil chemo attractant in agro gel first pipette, 10 milliliters of two times PBS and A 50 milliliter conical tube. Warm up the tube by putting it into a beaker containing hot water. Pipette 10 milliliters of distilled water.
Add 0.4 grams of aros powder and another 50 milliliter conical tube. Heat the mixture Until just boiling in a microwave oven to make 2%agros in PBS. Add the warm two times PBS to the agro solution tube.
Swirl the mixed solution and keep it warm in the hot water beaker. Mix well the chemo attractant solution with three microliters of India Ink in the lid of a 1.5 milliliter eend tube. Avoid generating an air bubble during the mixing.
Cut the tip end of a 200 microliter pipette tip and micro pipette. 110 microliters of agro solution at 42 degrees Celsius into the lid and immediately mix well. Using another pipette tip store this chemo attractant containing gel in the fridge until use Anesthetize An adult male mouse by an IP injection of a mixture of xylazine and ketamine.
Hydrochloride shave the area over the right external jugular vein and the anterior aspect of the scrotum. With an electric razor after anesthesia, it is important to give special attention and care to the anesthetized mouse. A heat lamp can be used to prevent the mouse from hypothermia.
The mouse should be free from pain Reflex. Make a Horizontal incision, find and catheterize the jugular vein using a PE 10 tubing filled with 100 units per milliliter heparin saline. Connect the board to a 37 degree Celsius water circulator to keep the cremaster muscle and the most body warm.
Fix mouse hind legs with umbilical tapes with the most lying face up on the cremaster muscle board. Make an incision in the scrotal skin to expose the left cremaster muscle. Carefully dissect the muscle from the associated fascia.
Super fused the cremaster muscle with 37 degrees Celsius warmed bicarbonate buffered saline. Tie a 4.0 suture in the distal end of the cremaster muscle to hold it down on the clear viewing glass pedestal of the cremaster muscle board. Cauterize the cremaster muscle longitudinally with a cautery with a four oh suture.
Hold the muscle flat and secure it along the edges on the pedestal. Separate the testicle and the epididymus from the underlying muscle and move them into the abdominal cavity. Add a thin layer of vacuum grease to opposite edges of a 22 by 22 millimeter glass cover slip.
Then cover the exposed muscle with the glass cover slip. Place the master muscle board on the microscope stage. Examine the muscle under the microscope and find a suitable post capillary venue.
Select the vennue that has a normal shear rate and a diameter in 25 to 40 micrometers. Adjust the video camera to allow the venue to be visualized in a vertical position on either the right or the left end of the TV monitor. After 30 minutes, record the video images of the selected post capillary venue as baseline control data using a video recorder device such as a DVD recorder.
This is the baseline control video images of the post capillary venue. Before placing The chemo attractant containing gel on the surface of the cremaster muscle, stop the superfusion and remove the glass cover slip on the muscle punch a one cubic millimeter size chemo attractant containing gel from the lid of the einor tube. Using a cut tip end of a 200 microliter pipette tip, place the punched chemo attractant containing gel on the surface of the cremaster muscle in a preselected area, 350 micrometers from the observed post capillary venue.
Add a cover slip to hold the gel in place and superf fuse the muscle tissue at a very slow rate, less than or equal to 10 microliters per minute to allow the establishment of a gradient of chemo attractant that is slowly released and formed from the gel. Record the video image for 90 minutes after the addition of the chemo attractant containing gel. During the recording, adjust and keep the microscope focus on the adhering, crawling, transmi migrating and chemo taxing leukocytes inside the venue and in the muscle tissue.
After the experiment, import the video file to a computer. For analysis on a computer, Extract and convert the video to a VI format. For example, use free computer software bit ripper to convert DVD video to an A VI file.
Use video editing software, for example, windows movie maker to make a time-lapse movie from the original realtime video convert and save the time-lapse movie to DV A VI format. Record the images of the calibration micrometer under the same microscope setting. Import images to the computer.
Open the images with image J.Measure the size of the screen at both x and Y axes and calculate the number of pixels per micrometer. To import the movie open image J.Again, click file import using QuickTime movies plugin, select the movie to be analyzed and click okay at the interface of QT movie opener, click plugins. Manual tracking to track cells fill in the relevant information into the fields at the bottom of the screen before starting tracking the following parameters should be determined before tracking time.
Interval in seconds is the full time lapse divided by 30 xy calibration is the micrometer per pixel measurement from the calibration of the image of the micrometer Z calibration is set at zero search square size for centering equals one. It is necessary to select in the recorded video a stable and clear point as a reference point. This reference point can be any clear and small structural point that remains unchanged and stable throughout the whole experiment.
Click add track to track the reference point from the first to the last frame. Then click and track track crawling and migrating neutrophils one by one. Click add track to track the cell from its appearance in the tissue to its disappearance in each frame.
Then click and track to finish and save the results in Microsoft Excel. Open the results file in Microsoft Excel. Start analyzing the data.
It is important to take the movements of the reference point into the analysis. When tracking neutrophil and luminal crawling, we determine the neutrophil and luminal crawling distance and crawling velocity. When analyzing neutrophil trans endothelial migration, we measure neutrophil trans migration time and the detachment time.
When determining neutrophil chemotaxis incre master muscle tissue, we measure the distance and velocity of neutrophil migration and chemotaxis in the muscle, and we also determine the chemotaxis index during neutrophil chemotaxis in muscle tissue. The definitions of these parameters shown on the screen are given in Detail in our written protocol. In this Experiment, we demonstrate the procedures of how to use bright field intra vital microscopic to visualize and determine the neutrophil intraluminal crawling T endothelial migration and migration in chemotaxis.
In response to neutrophil chemoattractant in vivo, we used either MIP two CX CL two, or synthetic peptide W-K-Y-M-V-M prepared an agro gel to induce neutrophil recruitment in mouse CMA muscle tissue. As we can see from this figure MIP two at 0.5 MICROMOLAR and W-K-Y-M-V-M at 0.1, millimolar elicited neutrophil intraluminal crawling at similar velocities. The neutrophil trans endothelial migration and the detachment from the ven were comparable in the length of time.
These two chemo attractants also induced neutrophil migration and chemotaxis in muscle tissue at nearly the same velocity and with similar neutrophil chemotaxis indexes. Intra vital microscope Is a valuable tool for direct observation of the process of neutrophil recruitment and for quantitative determination of the functions of cells and molecules in each recruitment step. By using time-lapse video photography and image J, the intraluminal crawling T endothelial migration and migration in chemotaxis of leukocytes and tissue can be measured under brightfield, intra vital, microscopic with the selective inhibitors, transgenic, mice, and selective chemo attractants.
The assay system can help us to reveal the functions of specific proteins in leukocyte recruitment.
