354 PAUL S. RUSSELL 



Intravenous injections 



Except where otherwise noted all injections were performed 

 through the facial or submental vein of the newborn mouse 

 using a no. 30 gauge needle according to the method of Billingham 

 and Brent (1957). No more than twelve hours were allowed to 

 elapse between birth and the initial spleen cell injection. Animals 

 dying within 48 hours after injection were assumed to have 

 succumbed to its direct mechanical consequences and were 

 excluded from further consideration. 



Skin grafting and sensitization 



In those experiments requiring spleen cells from donors 

 previously sensitized to the tissues of another mouse strain this 

 was accomplished by either of two different methods. The usual 

 method consisted merely of applying full-thickness skin homo- 

 grafts bilaterally to bare areas prepared on the thoracic wall as 

 described by Billingham and Medawar (195 1). The prolonged 

 state of sensitivity produced by rejection of such fixed tissue 

 grafts was usually reinforced in the animal not more than six days 

 before its use by an intraperitoneal injection of about 10 milhon 

 dissociated spleen cells derived from an adult member of the same 

 donor strain. 



The other method of sensitization made use of suspensions of 

 donor spleen cells exclusively as the antigen. The cell suspension, 

 prepared as described above, was mixed to form an emulsion in 

 equal volumes with a complete adjuvant mixture.* About ten 

 million cells were injected into each of the four foot pads of an 

 anaesthetized adult recipient on two different occasions seven 

 days apart. Five to nine days after the second dose of cells in 

 adjuvant, blood was collected by cardiac puncture and the serum 

 separated for use. The serum was used promptly without 



* Paraffin oil (Bayol F, Esso Standard Oil Co.), 8-5 ml.; mannide monooleate 

 (Arlacel A, Atlas Powder Co.), 1-5 ml.; heat-killed human tubercle bacilli, 

 40 mg. 



