Blood Harvest
・ Anesthetize mice with 50ml of Xylene-Ketamine(2:1) coctail
・ Pin animal to a board (use distal extremities) supine
・ Use 70% alcohol to moisten the abdomen and lower thorax
・ Make an midline incision using forceps and scissors from mid abdomen to xyphoid process
・ Dissect to the left side of the animal and open the peritoneum , exposing the liver and diaphragm
・ Lift the anterior chest wall with a forcep, the heart should be apparent though the translucent diaphragm
・ Fill a 1cc syringe with 300 ml of heparin
・ Holding the syringe comfortably, with one concert motion insert the needle into the heart through the diaphragm and draw blood
・ Should the first attempt not succeed and pneumothorax develops, immediately open the chest cavity through a midline incision (sternotomy) and draw blood directly from the heart
Labeling lymphocytes and separating lymphocytes from the whole blood
・ Add 250ml of DMEM with 10% Fetal Goat Serum(FGS) to 250ml of whole blood to block nonspecific binding
・ Incubate at 4°C for 10 minutes
・ Add 1mg of appropriate antibodies to the specimen (usually about a million cells)
・ Incubate at 4°C for 30 minutes
・ Add 2ml of cold lytic buffer solution to each specimen for 2 minutes � gently shake them
・ After 2 minutes fill the facs tubes with PBS
・ Spin at 1000rpm (450g) for 10 minutes at 4°C
・ Aspirate supernatant to pallet using vacuum setup
・ Add 2ml of lytic buffer solution for the second time for the residual RBCs and fill tubes with PBS. Spin again for 10 minutes at 1000rpm at 4°C
・ Aspirate supernatant with vacuum setup to pallet
・ Add ? ml of PBS to the pallets and gently mix them
・ Specimens are ready to be read by the facs machine. They can be stored at 4°C for at least 2 days.
