To begin, place the euthanized mouse in a supine position on the clean bench. Cut along the midline of the mouse abdomen and separate the abdominal structures layer by layer. Using tweezers, gently open the greater omentum and stomach.
Then gently pull out the spleen with the gastrosplenic ligament and bluntly separate it from surrounding tissues and ligaments to obtain an intact spleen. Place a 70 micron cell filter in a 100 millimeter sterile culture dish. Add the spleen to the cell strainer and crush it with a syringe plunger.
Add five to eight milliliters of homogenate flushing fluid to push the splenic cells through the filter into the culture dish. Centrifuge the filtrate at 450g for five minutes at room temperature and discard the supernatant. Re-suspend the pellet with the sample diluent provided with the lymphocyte separation kit.
After counting the cells on a hemocytometer, adjust the cell concentration to 2 X 10 to the power of eight cells per milliliter. In a centrifuge tube, take the same amount of lymphocyte separation solution as the tissue single cell suspension. Carefully pipette the single cell suspension onto the surface of the lymphocyte separation solution and centrifuge at 800g for 30 minutes at 25 degrees Celsius.
After centrifugation, observe the four layers in the tube from top to bottom. Using a pipette, carefully draw the annular milky white lymphocyte layer into another tube. Add 10 milliliters of cleaning solution to the tube to mix the cells.
Centrifuge the cell suspension at 250g for five minutes. Discard the supernatant and re-suspend the pellet in 10 milliliters of RPMI 1640 medium for cell counting.
