The overall goal of this procedure is to induce EAE consistently and easily in any mouse strain, including those reputed to be resistant to EAE induction. This is accomplished by first immunizing donor mice with myelin specific proteins or peptide antigens, emulsified in complete fres adjuvant. 10 days later, the lymph nodes from the donor mice are isolated and lymph node cells are re stimulated in vitro by culturing them with the same antigen for four to five days.
The cells are then adoptively transferred into naive recipient mice. Susceptible mouse strains will develop EAE in eight to 14 days to trigger EAE in a resistant strain. 14 days after the adoptive transfer, the recipient mice were then immunized with antigen in CFA.
Using this method, EAE can be induced in resistant mouse strains. The main advantage of the adaptive transfer and challenge protocol for inducing EAE is that it allows for reliable disease induction. Rarely do we see less than 100%clinical disease.
We can tailor the protocol for inducing disease and susceptible mouse strains, but more importantly, we can use this protocol to induce EAE in many mouse strains previously considered resistant to disease. Demonstrating the procedures. Today is our lead research associate, cha ching.
Begin by preparing a two milligram per milliliter solution of MBP. In PBS. Use a ground glass syringe equipped with a lure lock to draw up 100 microliters of antigen solution per mouse to be immunized.
Using a separate syringe, collect an equal volume of complete Freud's adjuvant H 37 ra. Then with a three-way stopcock, connect the two syringes and push the plungers back and forth to mix the antigen and adjuvant until an emulsion is formed. To verify that this is the case, transfer the solution to one of the syringes and disconnect the empty one.
Pull the empty syringes plunge back slightly, then discharge a drop of the residual mixture into a beaker of clean water at room temperature. If the drop disperses, reconnect the syringes and keep mixing. Repeat the test if the drop remains intact and emulsion has been formed to collect emulsion.
For immunizing. Connect a plastic one milliliter syringe to the glass syringe whose plunger has been removed. Carefully transfer the solution to the plastic syringe, filling it to the rim.
Then insert the plastic plunger and expel some emulsion so that exactly 1.2 milliliters remain. There should be no air bubbles trapped in the syringe. If this procedure is performed correctly.
Disconnect the plastic syringe. Fit it with a 26 gauge needle and fill the needle void volume by pushing the plunger to the one milliliter marking. Scruff the mouse firmly and hold the tail back between the ring finger and pinky so that the thorax and abdomen are easily accessible.Subcutaneously.
Inject 50 microliters of emulsion in the thorax right above the right armpit. If the injection is performed correctly, a little white bulge will be visible at the site of injection. Repeat the procedure above the left armpit, then release the tail and take hold of the right foot, exposing the right flank of the mouse.
Inject 50 microliters of solution just below the ribcage. Let go of the right foot. Turn the mouse the other way and repeat the injection on the left side.
Seven to 10 days following the immunization. Harvest the lymph nodes from euthanized animals. First place the mouse on its back with its limbs stretched out and secured to the dissection board with pins.
Spray the mouse with ethanol. Then using sterile tools. Cut a longitudinal incision in the mouse's skin from the lower abdomen up to the chin.
Make another incision from the abdomen down one leg. Then down the other. Peel the skin off the mouse and pin it to the dissection board.
Also cut the skin along the four limbs. Then peel off and secure the upper part of the skin flaps with pins on either side, the inguinal and the axillary lymph nodes, which are normally close to the groin or the armpits, should be visible as small opaque masses directly under the skin. Use two fine forceps to gently remove the connective tissue above the first lymph node.
Taking care not to disrupt blood vessels in the process. Once the connective tissue has been cleared, place one pair of forceps under the lymph node and pull it away from the body. Transfer the lymph node to a 35 millimeter Petri dish containing sterile PBS and harvest the other three lymph nodes the same way.
Once all the lymph nodes have been harvested, transferred the Petri dish to a biosafety hood. Using the back of a sterile syringe plunger. Homogenize the lymph nodes to liberate the cells to remove the debris, filter the suspension through a nylon mesh into a conical tube.
Then adjust the volume to 50 milliliters with sterile PBS and centrifuge the cells for 10 minutes. Following the centrifugation, resuspend the cells in a small volume of prewarm cell culture medium and count the viable cells. Adjust the concentration to four times 10 to the six cells per milliliter.
Then plate the cells at two milliliters per well in a 24 well plate. Add MVP antigen to each well so as to obtain a final concentration of 100 micrograms per milliliter. Once the antigen has been added.
Incubate the cells at 37 degrees Celsius for five days. Once the cells have been re stimulated, harvest them by gently pipetting up and down and transfer them to a 50 milliliter conical tube. Centrifuge the cells at 1000 RPM for 10 minutes and resuspend the pellet into a small volume of sterile PBS.
Then count the cells and adjust the viable cell concentration to 2.5 times 10 to the eighth cells per milliliter with PBS. Draw the cells up into a syringe and fit it with a 26 gauge needle to perform the adoptive transfer. Inject 0.2 milliliters of cells intravenously in the tail of each mouse to challenge resistant mice to weeks after the adoptive transfer.
Inject the animals with 200 micrograms of antigen in a volume of 200 microliters of emulsion the same way as the immunization of donor mice was performed. Seven to 10 days after the challenge, paralytic disease will develop and its progression can be monitored. Disease begins with weakness in the tail.
When the tail is flacid, the disease is graded, one disease is graded. Two, if the mouse develops paralysis in one hind limb, and three, if both hind limbs are paralyzed. Grade four is when both front limbs are paralyzed.
Grade five, the mouse is more abundant. And grade six, the mouse is dead. As shown here, seven to 14 days after challenge, mice will develop monophasic paralytic disease, which can be graded using the classical EAE criteria.
While the mice may recover, they will not spontaneously relapse. The induction of disease requires an antigen specific challenge. Challenge with CFA alone or an irrelevant antigen fails to elicit disease.
After watching this video, you should have a good understanding of the adoptive transfer and challenge method for inducing EAE. By combining adaptive transfer of antigen specific lymph node cells with a boosting antigenic challenge, one can easily induce EAE in any mouse strain. This method allows us to overcome the resistance mechanisms inherent in some mouse strains, and therefore allows the study of disease resistance.Also.
This technique should be easily adapted to study any autoimmune disease process.
