Imaging Transgenic Beta-Cells in an Immunodeficient Mouse Challenged with Diabetic Splenocytes

Begin with a restrained NOD immunodeficient mouse that lacks immune cells and is prone to diabetes.

This mouse is pre-injected with transgenic pancreatic beta-cells that produce insulin and express luciferase enzymes.

Take a syringe containing pre-treated splenocytes derived from a diabetic mouse. The splenocytes include autoreactive T cells with the ability to recognize the insulin peptides.

Administer these splenocytes into the mouse's tail vein.

The splenocytes circulate and reach the pancreas, where the autoreactive T cells recognize the insulin peptides on the beta cells and become activated.

These activated cells release cytotoxic molecules, initiating beta-cell destruction via autoreactivity.

Next, inject a luciferase substrate into the peritoneum of the mouse. The substrate diffuses in this region and enters the beta cells.

Within the cells, the substrate interacts with the luciferase enzymes, producing luminescence.

Using a bioluminescent imaging system, capture images at regular intervals

A gradual decrease in luminescence indicates the destruction of transgenic beta-cells due to autoreactivity.

Warm up the body of the adult recipient, NOD-scid mice by using a heat lamp for 5 to 10 minutes to vasodilate the veins. Resuspend the splenocytes' solution prior to each injection. For each mouse, draw 100 microliters of the pre-warmed splenocytes' 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 splenocytes' 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 5 minutes before imaging, inject the mouse intraperitoneally with the appropriate dosage of D-luciferin solution, using a 1-milliliter syringe and a 26-gauge needle.

After logging in to the imaging software, in the control panel select Initialize. To autosave the imaging data, click on Acquisition followed by Auto-Save 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.