144 D. W. H. Barnes and J. F. Loutit 



However, until an accredited cell-free material is shown to be 

 potent, or until cells from the donated material are shown 

 unequivocally to repopulate the host, there can be no absolute 

 confirmation of either hypothesis. 



Two sets of recent observations from the Radiobiological 

 Research Unit allow us now to be dogmatic that, unlikely as it 

 originally seemed, repopulation from the donor does in fact occur. 

 1. Mitchison (1956) used the inbred strains of mice, A and 

 CBA, with which our previous work had been done. CBA male 

 mice of about three months of age were irradiated as in 

 previous experiments with 950 r X-rays (240 kv. ; 15 mA.; 

 HVL =1-2 mm. Cu; dose rate 43 r/min.). This dose is almost 

 invariably lethal to untreated mice. On the same day, after 

 irradiation, the CBA mice were injected intravenously with a 

 suspension of cells from the spleen of infant mice of strain A. 

 The fresh spleens were cut into small pieces, suspended in 

 fresh rabbit serum and macerated with an electrically driven 

 mincer. Each mouse received 0-4 ml., the equivalent of 

 two-fifths of a spleen — that is about 15 X 10^ cells estimated 

 from counts made in a haemocytometer. These mice were 

 sacrificed at intervals and tissues were taken for test of their 

 content of antigens specific for strain A. 



The method of test involved administration of the respective 

 tissues to normal CBA mice. Each tissue for test was macer- 

 ated and injected intraperitoneally. If it contained, in 

 adequate numbers, cells derived from strain A it would thereby 

 induce in the normal CBA mouse, within a period of 8-12 

 days, the state of transplantation immunity. The injected 

 CBA mouse was, after the interval of 8-12 days, inoculated 

 subcutaneously with a suspension of tumour cells specific for 

 mice of strain A — sarcoma 1 (Dunham and Stewart, 1953). 

 This tumour had been maintained in the ascites form by 

 repeated passage in strain A mice. Ascites fluid was diluted 

 with isotonic sodium citrate until it contained approximately 

 10® cells in the 0-1 ml. used for injection. The reaction was 

 scored after a further 8 days when the tumour, or its remnant, 

 was carefully excised and weighed. 



