An Ex Vivo Technique to Induce Neuronal Death via Oligodendrocyte-Specific CD8+ T Cells
Transcrição
For this experiment, have 8- to 10-week-old transgenic ODC-OVA mice and age- and sex-matched C57Bl/6 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 vibratome plate, then, fill the plate with ice-cold placedine physiological saline. Once prepared, cut 300-micron slices. Transfer the slices to individual wells of a 12- or 24-well plate filled with aCSF. Then carefully add 1/2 a million OT-I T cells to each slice.
Half a million activated cells per slice provides good cell-cell interaction. Once 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 Celsius for further histological studies.
