Harvesting lymphocytes from Peripheral Blood

 

9.1.99

 

 

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 ½ 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.