MODIFICATION OF RUNT DISEASE 353 



genetically at the H-2 locus, C57BL/6 being classed as H-2^ 

 and DBA/ 1 as H-2^. This strain combination thus provides 

 major histocompatibility differences in both directions (Amos, 

 1959). 



When obviously pregnant the mother of each prospective 

 litter was placed in a separate cage where she was kept with her 

 young for the remainder of the experiment. 



Cell suspensions 



The donor spleens were promptly removed from adult DBA/ 1 

 mice with aseptic precautions immediately after sacrifice by 

 cervical dislocation. One to two spleens usually provided a 

 sufficient number of dissociated cells free from coimective tissue 

 for injecting a single litter since a harvest of about 500,000 

 usable nucleated cells per mg. of spleen was obtained. The 

 spleens were pooled in a sterile Petri dish, cleaned of surrounding 

 connective tissue, cut into small pieces with fme scissors, and 

 gently pressed once through a stainless steel sieve (250 holes/cm.^), 

 washing hberally with a balanced tissue culture medium 

 (TC-199)* as described by Billingham, Brent and Medawar 

 (1956). 



The cell clumps were broken up by gentle flushing through a 

 fairly coarse pipette and were concentrated in a plug by centri- 

 fuging at about 500 g for five minutes. The cells were then 

 washed twice more by resuspension in 6 to 8 ml. of TC-199 and 

 centrifuged as before, thus avoiding the deaths in acute apnoea 

 which have been known to follow the injection of appreciable 

 quantities of the fragments of broken cells (Billingham and 

 Brent, 1959). No anticoagulant was used. The fmal volume was 

 adjusted so that the prescribed number of nucleated cells, as 

 determined by haemocytometer counts, could be delivered in a 

 volume of o- 04 to o- 07 ml. 



* TC-199 available from Microbiological Associates, Bethesda, Maryland. 



