The overall goal of this intra vital confocal microscopy procedure is to monitor the dynamics of neutrophils and monocytes in mesenteric veins under steady state and inflammatory conditions in mice. This method can help answer key questions in the immunology field, such as what are the molecular basis of leukocyte migration, recruitment and interaction with the vascular endothelium. The main advantages of this technique are the ability to track and analyze the patrolling of LY 60 low monocytes under statist state conditions, and to follow the recruitment of both monocytes and neutrophils to the same vessel After local inflammatory stimuli demonstrating the procedure will be Stefan ler the technician from the laboratory To prepare a single cell preparation from bone marrow, sterilize the hind legs of an eight to 12 week old mouse with 70%ethanol, and then use a scalpel to harvest the femurs and tibia is when all of the bones have been disconnected.
Rinse the legs with 90%ethanol and transfer the bones into a culture dish filled with PBS. Then in a culture hood, use the scalpel to clean the femurs and tibia and to separate them at the knee joint. Next, cut off the ends of the bones and use a one milliliter syringe built with preparation medium equipped with a 23 gauge needle to flush the marrow from each bone into a 50 milliliter conical tube.
Disrupt the cell aggregates by passing them back through the needle tip, and then bring the total volume of the cell suspension up to 25 milliliters with fresh preparation, medium spin down the cells, then resus. Suspend the palate in one milliliter of red blood cell lysis buffer, and transfer the cells to a 15 milliliter conical tube on ice. After 30 seconds, stop the lysis with 10 milliliters of preparation.Medium.
Spin down the cells again and Resus suspend the pellet in. Two more milliliters of preparation medium to enrich the neutrophils by negative selection. First, incubate the bone marrow cells with 150 microliters of mouse neutrophil enrichment cocktail on ice for 10 minutes.
At the end of the incubation at 10 milliliters of phenol red free dmem. Invert the tube one time and spin down the cells. Re suspend the pellet in 1.75 milliliters of preparation medium, and transfer the cells into a five milliliter polystyrene round bottom tube.
Then incubate the cells with 250 microliters of biotin selection cocktail on ice for 10 minutes. At the end of the incubation, we suspend the magnetic particles from the cell isolation kit with 30 seconds of vortexing. Then incubate the cells with 500 microliters of the particles on ice for 10 minutes.
At the end of the incubation, place the tube into the magnet. After three minutes, invert the magnet into a new five milliliter polystyrene round bottom tube. To collect the unlabeled cells, place the tube of unlabeled cells into the magnet.
After three minutes, invert the magnet into a 15 milliliter conical tube to transfer the unlabeled cells leaving the last drop in the tube. Then bring the volume up to five milliliters with fresh phenol red free dm. Now add half a microliter of the appropriate cytoplasmic dye such as the cell tracker orange used here to the cells at 37 degrees Celsius.
After two minutes, a 37 degrees Celsius, wash the cells with 2.5 milliliters of preparation medium and resuspend the pellet in one milliliter of preparation medium in a 1.5 milliliter tube, count the cells and then incubate them for five minutes at 37 degrees Celsius. After spinning down the cells, we suspend the pellet in one milliliter of PBS followed by another centrifugation while the cells are spinning. Use oil to attach a 35 millimeter glass cover slip to a 10 centimeter tissue culture dish with a 30 millimeter diameter hole.
Then resuspend the pellet at one times 10 to the seven cells per 100 microliters of PBS and place the cells on ice as soon after labeling as possible. Inject the labeled neutrophil cells intravenously into an anesthetized eight to 12 week old CX three CR one GFP mouse, and place the mouse on a heating pad. Then immediately apply ointment to the animal's eyes and use scissors to open the abdominal skin, exposing the peritoneum, incise the peritoneum with the scissors to expose the intestine.
Then administer 200 microliters of 37 degrees Celsius PBS to the peritoneal cavity and turn the mouse face down over the tissue culture dish to remove the intestines from the peritoneal cavity with gentle pressure. Then using sterile cotton buds carefully droll the intestines onto the cover slip to expose the mesenteric vessels and immobilize the tissue with pieces of tissue paper wet with 37 degrees Celsius PBS to decrease the movements resulting from peristalsis. It's very important to take your time to carefully immobilize the intestine with PBS weighted tissues.
Otherwise movements resulting from peristaltic during image acquisition will ruin the experiment. Next, transfer the tissue culture dish and mouse into a 37 degree Celsius thermostat chamber on a custom made trace stage of an inverted microscope and administer a second round of anesthetics intramuscularly to the hind leg. Now set the appropriate lasers to the lowest power necessary to avoid laser induced phototoxicity and allow the mouse to rest for 30 minutes.
When the animal has recovered, record the first movie for 30 minutes under steady state conditions. Then to initiate the blood vessel inflammation, dispense a 20 microliter drop of PBS containing 100 micrograms of the TLR two TLR one agonist PAM three CSK four directly onto the imaged vessels. Finally acquire a second movie taking care to control the focus after addition of the agonist as the inflammation induces changes in the endothelium width, altering the focus of the Z axis here, the different steps of image processing for the first time point of a movie acquired under steady state conditions, as just demonstrated, are shown in this movie.
The patrolling of green LASIK cilo monocytes under steady state conditions can be visualized with red neutrophils observed circulating in the bloodstream.Here. The same field of interest is shown 90 minutes after the addition of PAM three CSK four, resulting in the massive recruitment of neutrophils and monocytes to the endothelial wall, which the monocytes scan meticulously using the appropriate software, the exact paths of the monocytes and neutrophils. Patrolling the endothelium can be tracked before and after the initiation of the inflammation.
It is important to note that a loss of focus on the Z axis or an XY displacement can occur as a consequence of intestinal movements ruining the experiment. This technique can be further modified by using different genetically modified mice models or injecting blocking antibodies or pharmaceutical drugs to determine the role of specific molecules of interest in leukocyte behavior. After watching this video, you should have a good understanding of how to monitor the dynamics of saturating leukocytes in mesenteric veins.
