An Ex vivo Model of an Oligodendrocyte-directed T-Cell Attack in Acute Brain Slices

The overall goal of this procedure is to study an oligodendrocyte directed attack of CD eight T cells ex vivo. This is accomplished by first isolating CD eight T cells from the spleens of mice. The second step is to prepare acute brain slices.

Then the brain slices are incubated with the activated CD eight T cells for up to six hours, followed by cryo conservation and histological studies. Ultimately, immunofluorescence microscopy can be used to study the location of T cells as well as apoptosis of oligodendrocytes. The main advantage of this method over existing techniques like experimental autoimmune encephalomyelitis, an animal model of milds sclerosis is that neuron damage.

Following CD eight mediated oligodendrocyte attack can be studied in detail. This method can help answer key questions in the field of neuroimmunology, such as immune cell trafficking in tissue, or degeneration of neurons and oligodendrocytes. Demonstrating the procedure will be Alexander Hamman, a grad student from my lab Five days before preparing the brain slices stimulate the OT one T cells to activate the cells.

First, follow the sighted protocol to remove the spleens of several O OT one transgenic mice. Collect the spleens in freshly prepared, supplemented medium. Next, create a single cell suspension by mashing the spleens through a 70 micron strainer, and then centrifuge the suspension at 300 Gs for five minutes at four degrees Celsius.

Following the centrifugation, pour off the supernatant and resuspend the cells in five milliliters of ammonium chloride potassium buffer. Let the cells incubate in this buffer for five minutes at four degrees Celsius to lyse the red blood cells. Then end the reaction by adding 10 milliliters of supplemented media at four degrees Celsius.

Next, collect the cells again by spinning them down as before. Then collect the pelleted cells, resus, suspend them in media and count them when their density is known. Transfer the cells to a 12 Well plate at 30 million cells per well.

Then add media with OVA and interleukin two and incubate the cells for four days. After four days, add more interleukin two at a concentration of 500 units per milliliter. Then incubate the cells for another day.

Now using a CD eight positive T cell isolation kit, purify the O OT one T cells from the cultured cells.Briefly. Non CD eight positive cells are depleted by using a cocktail of antibodies and are separated using a magnetic column. In doing this, prepare the sample on ice and make sure that the reaction involves only single cells, so the column is not blocked.

Then count the cells. For this experiment. Have eight to 10 week old transgenic, O-D-C-O-V-A, mice and age and sex matched C 57 black six controls.

As usual, house the mice in a pathogen free environment with free access to food and water. After anesthetizing a mouse, use a back paw pinch to assess the level of anesthesia and then immediately euthanize it. Now position a mouse ventrally and disinfect the scalp and decapitate it.

Next quickly, cut the scalp, open the cranium and scoop out the brain. Fix the brain in place using glue and transfer it to a vibram plate. Then fill the plate with ice cold ine physiological saline.

Once prepared, cut 300 micron slices. Transfer the slices to individual wells of a 12 or 24 well plate filled with A CSF. Then carefully add half a million OT one T cells to each slice.

Half a million activated cells Personalized provides good cell cell interaction. Once cells, the cells start migrating into the slice. At the same time, the amount of cells is not too high to induce an overreaction, so single cell counting is feasible.

After loading all the wells, incubate the co-culture for up to eight hours after the incubation, harvest the slices and embed them in OCT compound. Then freeze the embedded slices in liquid nitrogen and store them at negative 20 degrees Celsius for further histological studies. After incubation of brain slices with oligodendrocyte directed CD eight positive T cells, oligodendrocytes underwent apoptosis.

Neurons also underwent apoptosis. The earliest signs of apoptosis were seen after three hours of incubation and were robust by eight hours. Apoptosis was more noted in O-D-C-O-V-A cells exposed to OT one T cells than in any other condition.

Following this procedure, additional methods can be applied to answer questions concerning T-cell migratory behavior, or the involvement of ion channels in your own lab ptosis. After watching this video, you should have a good understanding of how to prepare acute brain slices and how to co-culture them with T cells.