TRANSPLANTATION ANTIGENS AND HAEMAGGLUTININS 27 



The spleens collected, with all required aseptic precautions, are 

 gently chopped and expressed through a stainless steel sieve into 

 buffered normal saline at o°c. The cells isolated by vigorous 

 pipetting are counted, disregarding the red cells. 



Splenic extracts are prepared by two different techniques: 



(i) the technique of Billingham, Brent and Medawar (1958), 

 where the cells are brought into solution in water by a short 

 exposure to ultrasonic irradiation, DNA proteins being pre- 

 cipitated by the addition of NaCl to o* 15-M strength and removed 

 by centrifugation at 2,000^. The supernatant is finally spun at 

 25,000^ for one hour. 



(2) the technique of Oth and Caster mans (1959) by which the 

 extraction is obtained by homogenization in a Waring blendor in 

 an ice-cold solution of 0*14 M-NaCl, o*ooi-m sodium citrate, 

 and a secondary acid precipitation at pH 5*5. 



Epidermal cells are prepared according to a previously described 

 method (Albert and Lejeune-Ledant, 1959). Skin taken from the 

 tails of the mice is collected in a buffered solution and submitted 

 to the action of trypsin for 20 minutes at 37°. The epidermal sheets 

 are expressed through the stainless steel sieve, and the cells isolated 

 by pipetting. These epidermal cells are always injected intra- 

 venously, the only route we have found effective for induction of 

 a transplantation immunity (Lejeune-Ledant, i960). 



Epidermal extracts (Lejeune-Ledant and Albert, i960) are 

 obtained by digestion of the isolated cells submitted to the action 

 of a 0-25 mg. per cent concentration of trypsin in a buffered 

 solution: 0-137 M-NaCl, 0-0026 M-KCl, 0-0081 M-Na2HP04, 

 0-00147 M-KH2PO4, 0-0009 M-CaCl2 and 0-00048 M-MgClg. 

 6H2O. Ca and Mg ions are intended to protect the antigens 

 against the action of trypsin. After incubating from 5 to 20 

 minutes at 37°, all the subsequent manipulations are performed 

 at +4°. Trypsin is eUminated by repeated washings in 0-15 

 M-NaCl and centrifugation at 1500^. The final antigenic extract 

 is suspended in pure water. 



