This is a cached page for the URL (http://www.surgeryresearch.com/Protocols/UM/Peripheral%20Lymphocyte%20Harvesting.htm). To see the most recent version of this page, please click here.
Protocol Online is not affiliated with the authors of this page nor responsible for its content.
About Cache
Harvesting lymphocytes from Murine Liver

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 4C for 10 minutes

        Add 1mg of appropriate antibodies to the specimen (usually about a million cells)

        Incubate at 4C 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 4C

        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 4C

        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 4C for at least 2 days.