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        Harvesting lymphocytes from Peripheral Blood

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        601

         

        Blood Harvest

         

                Anesthetize mice with 50 m l 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 m l 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 250 m l of DMEM with 10% Fetal Goat Serum(FGS) to 250 m l of whole blood to block nonspecific binding

         

                Incubate at 4 ° C for 10 minutes

         

                Add 1 m g 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.

         

         

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