The aim of this procedure is to study T-cell trafficking and accumulation patterns during graft versus host disease. This is accomplished by first isolating bone marrow and cytes from donor mice. The second step is to purify cell populations of interest and prepare them for transplantation.
Next, the purified cell populations are combined and transplanted into a radiated recipient mice. The final step is to visualize T cell trafficking and accumulation patterns in recipient mice. Ultimately, bioluminescent imaging is used to show T-cell trafficking and accumulation patterns during GVHD.
The main advantage of bioluminescent imaging over existing technologies is it allows a means to detect lymphocytes throughout the entire mouse as opposed to at a specific site as as with existing technologies Recipient. Mice must be treated with lethal irradiation prior to bone marrow and cyte transplantation. Place up to 10 recipient mice in a micro isolator cage compatible with the irradiator to be used.
Next, irradiate the mice with either a cesium 1 37 source or a RS 2000, a radiator with an initial dose of 4.5 centigrade. Three hours later, irradiate the mice again with 4.5 centigrades for a total of nine centigrades of radiation. After the second dose store the recipient mice in a micro isolator cage with acidified water with a pH of three until the time of transplant.
Acidified water will help control opportunistic affections in recipients to obtain PLE cytes. Cut through the skin of a euthanized donor mouse by making a vertical two centimeter incision about two centimeters right of the midline directly under the rib cage in order to localize the spleen. Then remove the spleen and place it in a 40 microliter mesh green in a Petri dish containing 1640 RPMI with 5%FBS.
To prepare single cell suspensions, use a syringe plunger to break the spleen apart until the entire spleen has been passed through the mesh screen. Repeat this process for each wild type and luxe positive donor mouse. Collect all the wild type single cell suspensions in one 50 milliliter conical tube, and collect all the luxe positive single cell suspensions in a separate 50 milliliter conical tube.
Once the suspensions have been collected in their respective tubes, centrifuge the cells and resp. Suspend the pellets in 1640 RPMI with 5%FBS. Then count the cells.
Remove the skin from one hind limb of a donor mouse and carefully cut away as much muscle tissue as possible from the femur and tibia fibula using sturdy scissors. Cut the femur off at the hip joint to remove the hind limb. Then cut off the rear pore just below the tibia fibula insertion.
Carefully remove any remaining muscle tissue and cut away the fibula. There is little marrow in the fibula so it can be discarded. Next place the hind limb in cold 1640 RPMI with 5%FBS solution and repeat the process for the second hind limb.
Store hind limbs in media until all hind limbs are removed. Each mouse should yield between 20 times 10 to the six to 40 times 10 to the six bone marrow cells. To prepare for bone marrow removal, take one hind limb from the cold R-P-M-I-F-B-S media and place it in a large Petri dish containing one milliliter of cold media.
Cut off the knee joints from each hind limb. Then attach a 27 to 29 gauge subcutaneous needle to a 10 milliliter syringe and place the needle into the tibia. Depress the syringe until all of the red material is removed from the interior of the tibia.
Repeat this process with the femur. Discard any remaining bones which are devoid of bone marrow. Repeat this process with the second hind limb.
Pipette the media containing bone marrow into a large Petri dish. In order to prepare the single cell suspensions using a syringe, plunger and 40 microliter mesh screen, break the marrow apart until it completely passes through the screen. Next, pipette the single cell suspension into a 50 milliliter conical tube and place this tube on ice deplete CD three positive cells from the bone marrow following manufacturer's protocol.
Once the marrow is depleted of CD three positive cells wash the remaining cells by centrifuging the SP cytes and bone marrow cells do not combine cytes and bone marrow cells. Count the cells using a hemo cytometer. Next, remove all of the supinate Reese.
Suspend the bone marrow cells in 100 microliters of max buffer per 10 million bone marrow cells. Continue with CD three depletion using the manufacturer's protocol. Next, wash the CD three depleted bone marrow cells three times in sterile PBS.
Count the cells and resuspend them as an appropriate volume in order to inject 10 to the seven cells. Resus suspend the wild type pellet in 90 microliters of max buffer per 10 to the seven cells. Continue with CD eight T-cell depletion.
According to manufacturer's protocol, resuspend the luxe positive pellet in 40 microliters of max buffer per 10 to the seven cells. Continue with CD eight T-cell purification. According to manufacturer's protocol, count each cell population and wash each population three times with one milliliter of sterile PBS resuspend each population in an appropriate volume to inject 18 times 10 to the six cd, eight depleted wild type PLE cytes and two times 10 to the six L two G 85 B six purified CD eight T cells to prepare for injection.
First, combine 10 of the seven CD three depleted bone marrow cells 18 times 10 to the six while type CD eight depleted cytes, and two times 10 to the six L two G 85 B six lux positive purified CD eight T cells in a micro centrifuge tube. Then wash the combined cells with sterile PBS and Resus. Suspend them in 300 microliters of sterile PBS.
Next, using a 28 gauge needle, inject the cell preparation into the tail vein of a recipient mouse. Store the injected recipient mice in a micro isolator cage with acidified water. Score the mice daily using the GVHD scoring system shown in table one.
After injecting the mice with luciferian and imaging the anesthetized mice for five minutes at low benning, analyze the data using living image software. Regions of interest can be created using living image software and light emittance can be quantified by calculating the flux being emitted from each region of interest. Here is the GVHD scoring system developed by Cook Etol in 1996.
Mice should be scored daily on each criterion on the left. Each mouse is given a score of zero to two for each criteria and the total score is the sum of all individual scores shown. Here are clinical scores and survival data for the recipient mice who have been transplanted with 10 to the seven bone marrow cells alone or with 18 times 10 to the six CD eight T cell depleted wildtype cytes, and two times 10 to the six purified loose positive CD eight T cells.
Here the clinical score data for the recipients of bone marrow alone or with CD eight T depleted wildtype cytes and purified loose positive CD eight T-cell are shown the survival data of the recipients of bone marrow alone or recipients with CD eight T-cell depleted wildtype BLE cytes and loose positive CD eight T cells is shown here. These are bioluminescent images of recipient mice after injection with four milligrams D Lucifer in and imaged with Enogen IVIS for five minutes at small spinning. The pseudo colored images are shown where purple represents low intensity and red represents high intensity.
Regions of interest were drawn around the entire mouse and total flux or quantified. Shown here is a bioluminescence image of recipient mice after injection with four milligrams d Lucifer, and imaged with the Enogen IVIS for five minutes at small spinning. As in the previous image, the pseudo colored images are shown where purple represents low intensity and red represents high intensity panels.
A, B, and C represent imaging on days four, six, and eight post transfer respectively. Following this procedure, other methods like flow cytometry can be used to further characterize T cells that infiltrate the gut.
