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Harvesting lymphocytes from Peripheral Blood

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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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