To begin, position the euthanized mouse on a surgical platform and open the chest cavity. Using surgical scissors, make a transverse incision below the rib cage and cut ribs on each side of the mouse to the clavicle. Then cut the diaphragm to release the chest wall And lift ribs with forceps to expose the thoracic cavity.
Hold the thymus with forceps, and gently cut the connective tissue holding the thymus in place. To perform enzymatic dissociation of murine thymic tissue, place the cleaned thymus in a 15 milliliter tube cap and use sterile surgical scissors to finely mince the tissue. Add two milliliters of murine dissociation cocktail to the tube.
Attach the cap containing the thymus pieces and invert the tube five times to re-suspend the tissue. Wrap the tube cap in paraffin film. After incubation, place the tube in a rack and allow the tissue to settle.
Remove the supernatant and pass it over a 70 micrometer cell strainer into a 50 milliliter ice cold tube. Then, add 20 milliliters of M1 99 with 2%FBS medium to the cell suspension. Centrifuge the tube at 500 G for five minutes at four degrees Celsius.
After discarding the supernatant, re-suspend the pellet in M1 99 and 2%FBS medium.
