This protocol can help to understand the mechanisms of protection against autoimmune attacks and identify therapeutic targets for beta cell replacement in type one diabetes. The main advantage of this technique is the relatively low cost, the simplicity of the cell culture, transplant, and imaging methods, as well as minimal invasiveness of the mouse techniques. Individuals who have never performed a tail vein injection before may struggle to insert the needle into the vein.
Ask for a demonstration by an expert before your first injection. To begin, take the anesthetized mouse and remove hair from its dorsal side using an electric shaver with a guard, exposing approximately one inch by two inches of the skin. Return the shaved mouse to the knockdown chamber.
Before the transplant, transfer one mouse at a time from the chamber to a clean surface equipped with an isoflurane nose cone and gently guide the head of the mouse into the nose cone. Wipe the shaved area with an isopropyl alcohol prep pad to remove loose hair and disinfect the skin. Next, draw more than 300 microliters of cell suspension into a one milliliter sterile syringe fitted with a 26 gauge needle.
Remove any bubbles from the syringe and retain 300 microliters of cell suspension while pushing out the excess. Using a pair of curved forceps held in the non-dominant hand, gently lift the skin on one side of the mouse's back to allow easier access to the subcutaneous space. Then using the dominant hand, position the syringe parallel to the coronal and sagittal plains of the mouse's body.
With the needle pointed toward the head of the mouse, insert it into the skin near the hind quarters and guide it into the subcutaneous space while ensuring the entire needle remains under the skin and does not poke through. Adjust the forceps to hold the skin around the base of the needle. Slowly dispense less than 50 microliters of the cell suspension and confirm the formation of a small bulge beneath the skin at the site of the injection.
Continue injecting up to 200 microliters of the cell suspension subcutaneously. Then holding the forceps in place and keeping the needle parallel to the mouse's body, withdraw the needle. Once the needle is removed, hold the skin closed with forceps for a few seconds to prevent the cells from leaking out of the puncture wound.
Following the transplant, transfer each mouse to a fresh cage and allow the animal to fully recover from the anesthesia before adding additional mice to the cage. Placing the euthanized mouse on its back, use surgical scissors to make an approximately two inches long vertical incision in the skin. Cut open the right side of the mouse and locate the bright red spleen.
Gently cut the spleen away from the pink pancreas and transfer it to a sterile 10 centimeter Petri dish containing five milliliters of sterile PBS. Mash the spleen with the flat top of a sterile syringe plunger. Place a 40 or 70 micron strainer on a 50 milliliter conical tube and prime it with five milliliters of PBS.
Transfer the spleen suspension into the strainer and mash it gently. Wash the dish with 10 milliliters of PBS and transfer the wash to the strainer. Continue mashing until no red color remains on the strainer.
Then spin the tube at a speed of 500 G for five minutes at room temperature and remove the supernatant with an aspirating pipette before resuspending the cell pellet in five milliliters of ACK lysis buffer pre-warmed to room temperature to perform lysis of the red blood cells at room temperature for four minutes. Quench the reaction with five milliliters of NIT-1 cell media per five milliliters of lysis buffer and pass the cell suspension into a 50 milliliter centrifuge tube through a fresh strainer to remove clumps. Spin the tube at 500 G for five minutes at room temperature.
Next, resuspend the cell pellet in 20 milliliters of PBS before counting the cells. After counting, spin down the cells at 500 G for five minutes at room temperature. Resuspend the cell pellet in sterile PBS in a 1.5 milliliter safe-lock reaction tube.
Either inject immediately or store the cells on ice for a maximum of one hour. Warm up the body of the adult recipient NOD SCID mice by using a heat lamp for five to 10 minutes to vasodilate the veins. Resuspend the splenocyte solution prior to each injection.
For each mouse, draw 100 microliters of the pre-warmed splenocyte suspension into the syringe, ensure that no air bubbles are present. Next, place the mouse in the restraint device before capturing its tail with the non-dominant hand. Locate one of the two lateral tail veins.
Gently rotate the tail if needed. Wipe the tail with a disinfection wipe containing 70%isopropyl alcohol. Using the dominant hand, insert the needle at an acute angle into the central region of the tail.
With the bevel of the needle facing up, slide the needle a few millimeters through the skin. Then apply gentle pressure to the syringe to inject the splenocyte suspension. Do not allow the needle to move further in or out when injecting.
Successful injections do not give rise to back pressure during injection and are indicated by transparent or white blood flow immediately following injection. After injection, gently release the needle out of the vein and apply light pressure to the tail with a disinfection wipe until the bleeding stops. Release the mouse from the restraint device and gently transfer it to a freshly prepared cage.
At least five minutes before imaging, inject the mouse intraperitoneally with the appropriate dosage of D-luciferin solution using a one milliliter syringe and a 26 gauge needle. After logging into the imaging software, in the control panel, select initialize. To autosave the imaging data, click on acquisition followed by autosave to and create the appropriate folders in the computer.
Once the instrument has finished initializing, set the exposure time to one minute. Place the mouse on the imaging instrument in a prone position with its limbs splayed and guide its head into the nose cone. Flatten the mouse gently by pressing the center of its back with both hands and then spreading the hands outward and apart.
Then in the imaging software, select acquire and record any relevant details of the experiment in the popup window. In two out of the three mice studied, the control graft was destroyed by day 18 as evident from the loss of the respective bioluminescent signals. In the other mouse, however, the mutant graft was destroyed while the control graft survived, highlighting the biological variation between animals.
The bioluminescent signal was quantified and graft survival was reported as the percentage of the residual bioluminescent signal compared to the signal at the first time point. The ratio of luminescent signals from the mutant graft to the control graft for each mouse was used to visualize the variation between animals. Before disease transfer, the transplanted cells may replicate resulting in expanded grafts visible through the skin.
Upon imaging, these grafts displayed saturated bioluminescent signals which could not be accurately quantified. Grafts can be isolated at various time points for characterization of the infiltrating immune cells by, for example, flow cytometry to investigate the mechanisms of autoimmune attack and graft protection.
