The protocol we present here will be helpful for us to understand how lymphocytes are involved in the development of the central nervous system autoimmune disease. With this method, lymphocytes in the brain can be studied studied on a cell by cell basis. This protocol can also be applied to other models and central nervous system disease that needed to analyze the characteristics and functions of immune cells.
In this video, the protocol can easily demonstrate how to induce model and isolate single cells in the brain. After confirming a lack of response to pedal reflex, use a one milliliter syringe to subcutaneously inject anesthetized C57BL/6 mice with 100 micrograms of emulsified MOG 35-55 in 100 microliters of complete Freund's adjuvant into two different sites of the back near the neck. On the same day and on day two postimmunization, intraperitoneally inject the mice with 200 microliters of one nanogram per microliter pertussis toxin working solution.
After the second injection, transfer the mice back to their home cage with a warming pad and monitoring until full recumbency. The next day and throughout the course of the disease, examine and grade all of the mice in a blinded manner for the neurological signs outlined in the table and for any changes in weight. At the appropriate experimental endpoint, cut the cranium carefully from the nose to the neck and transfer the brain of each animal from the cranial box into individual 50 milliliter tubes containing 10 milliliters of RPMI medium.
Mix the tubes well to remove the adherent red blood cells and remove the medium by aspiration. Add 10 milliliters of fresh medium to each tube and transfer the brains into individual 100 millimeter dishes. Finally chop each brain with a razor and use a plastic pipette to transfer as much tissue from one dish at a time and six milliliters of medium into an icecold seven milliliter centered glass homogenizer.
Grind the brain using the loose plunger of the pestle before using the tight plunger to bring the tissue until the suspension is homogeneous. Then, pour the resulting tissue slurry into a pre-chilled 15 milliliter conical tube on ice. When all the samples have been homogenized, adjust the volume in each tube to seven milliliters with fresh medium, before adding each tissue suspension to a new chilled 15 milliliter tube containing three milliliters of 100%basement membrane matrix per tube.
Mix by inversion a few times and use a three milliliter pipette to carefully and slowly add one milliliter of 70 percent density gradient solution under each tissue solution sample. Separate the cells by density gradient centrifugation and remove almost all of the top layer, taking care to completely remove all of the myelin. Transfer the interface into a new 15 milliliter tube and adjust the volume to 10 milliliters with fresh medium.
Then centrifuge the samples again and remove the supernatant before resuspending the pellets in about 100 microliters of medium per tube. For flow cytometric analysis of the harvested brain cells, allocate approximately two times 10 to the six cells in 100 microliters of medium into individual wells of a 96-well plate. When all of the cells have been plated, add 100 microliters of 500X cell stimulation cocktail plus protein transport inhibitors to the wells and place the plate in the cell culture incubator for four hours.
At the end of the incubation pool the cells at the bottom of the wells by centrifugation and resuspend the pellets in 100 microliters of flow cytometry buffer per well. Pre-incubate the cells with three microliters of Anti-Mouse CD16/CD32 Fc Block for 10 minutes at four degrees Celsius before staining. To block any nonspecific Fc-mediated interactions.
At the end of the incubation, add the antibody cocktail of interest to each well and place the plate at four degrees Celsius protected from light. After 30 minutes, wash the wells with 100 microliters of flow cytometry buffer per well and sediment the cells by centrifugation. After discarding the supernatants, add 200 microliters of intracellular fixation buffer to each well.
After a 30 to 60 minute incubation at room temperature, collect the samples by centrifugation and resuspend the pellets in 200 microliters of 1X permeability buffer per well. Centrifuge the samples again, and after discarding the supernatant, resuspend the pellets in 100 microliters with fresh permeabilization buffer. Next, add the appropriate antibodies for the detection of any intracellular antigens of interest for a 30 minute incubation at four degrees Celsius, protected from light.
At the end of the incubation, add 100 microliters of 1X permeabilization buffer to each well and spin down the samples by centrifugation. Then, resuspend the stain cells in 200 microliters of flow cytometry staining buffer and analyze the cells by flow cytometry according to standard protocols. A typical clinical course of EAE should result in a disease curve and change in body weight as illustrated.
C57BL/6 mice immunized with MOG 35-55 usually start to develop disease symptoms around day 10 to 12 and achieve the peak of disease around day 15 to 21. Before the onset of disease, the body weight of immunized mice gradually increases before decreasing in correlation with the increasing disease symptoms The mice demonstrate the lowest body weight at the peak of EAE, with body weights recovering slightly as the clinical symptoms decrease. The mice usually do not fully recover however and typically go on to develop a monophasic chronic disease pathology.
As this representative flow analysis illustrates, interferon gamma-producing Th1 and IL17-producing Th17 cells are significantly increased in EAE mice. The most important step is 3.10. The operator should transfer middle layer and the light, being carefully, take as few other layers as possible.
Following these protocol, we can further T lymphocytes for further transcription analysis to study
