238 HILDEMANN, LINSCOTT AND MORLINO 



Materials and methods 



Separation of lymphocytes from the granulocytes and mono- 

 cytes of mouse blood was not easily accomplished. Initially we 

 tried the procedures of Jago (1956) and Cassen, Hitt and Hays 

 (1958) as applied to human blood, and a number of modifications, 

 including the method Terasaki (1959) successfully employed with 

 chicken blood. None of these techniques yielded concentrated 

 preparations of mouse lymphocytes with a high degree of purity 

 and viability. After much experimentation, the following pro- 

 cedure proved efficacious. Adult mice were lightly anaesthetized 

 with ether and blood was collected from a pocket cut between the 

 skin and thoracic wall after severing the subclavian artery. 

 About 7-8 ml. blood was mixed with o- 01 ml. of heparin sodium 

 (1,000 U.S.P. units/ ml.) in a calibrated centrifuge tube. This 

 blood was filtered through a glass wool column previously 

 prepared as follows: 



Wash Pyrex glass wool with hot running water, rinse with 

 distilled water and dry. Make a small hole in the bottom of a 

 Wassermann tube; pack 1-5 to i-8 cm. of glass wool in the 

 bottom of the tube as tightly as possible and autoclave. This 

 setup is for C57BL blood. Different lines of mice require different 

 amounts of glass wool for optimum yield and purity of 

 lymphocytes. 



The glass wool removed most, but not all, of the granulocytes 

 and monocytes from the blood which was collected in a sterile 

 Wassermann tube. This preparation was mixed and centrifuged 

 at 60^ for 10 minutes, followed by 500^ for 15 minutes. A long 

 Pasteur pipette with a narrowed orifice (sufficient to let a small 

 rubber bulb fill with air in 2-3 seconds after it has been squeezed) 

 was used and the buffy coat was carefully removed, care being 

 taken not to aspirate more than the uppermost layer of red cells 

 along with the lymphocytes. The above manipulations should be 

 performed at 20-25° c; good separations were not achieved at 



