110 ALEXANDER L. DOUNCE 



homogenizer was used for breaking the cells, and care was taken to keep the nuclear 

 suspensions quite dilute, especially in the last stages of purification, in order to over- 

 come agglutination of the mitochondria. These nuclei formed gels with alkali. No 

 work on enzyme content has yet been done. Liver cell nuclei also have been isolated 

 on a larger scale in 0.9% NaCl to which CaCU had been added. '' The latter nuclei 

 contained a higher percentage of DNA than nuclei isolated in very dilute citric acid 

 solution at pH 6.0. This is not surprising, since physiological saline solution extracts 

 protein from these nuclei as well as from the sucrose-calcium chloride nuclei pre- 

 pared by the more recent procedure. Nuclei prepared by the latest published proce- 

 dure of Hogeboom and Schneider" also lose protein when extracted with 0.9% NaCl 

 solution. In general, buffers or saline solutions tend to extract protein from cell 

 nuclei, and in addition they often tend to increase the resistance of whole cells to 

 breakage. 



A number of methods have been outlined above for obtaining cell nuclei in aqueous 

 media. These methods all have been applied to liver cells, but they are not all uni- 

 versally applicable. Kidney and pancreas are reasonably similar to liver in behavior, 

 but thymus differs. The original method for isolating cell nuclei at pH 6 in very dilute 

 citric acid is applicable to liver, kidney, and pancreas provided that steps are taken 

 to remove fiber where necessary, but generally it will not work with thymus. It is 

 quite likely that the calcium chloride-sucrose method discussed above would be as 

 general in application as any method involving the use of aqueous media, although 

 the reviewer has not yet had the opportunity to demonstrate the truth of this state- 

 ment. However Schneider and Peterman were able to use their original procedure'^ 

 to obtain nuclei from spleen and certain tumors, as well as from liver. Here, a high- 

 speed mixer was used to break the cells. 



In general it is very difficult to obtain clean nuclei from tumor cells by procedures 

 involving the use of aqueous media except by the use of strong citric acid (2 to 5%) 

 (see footnote'). A certain proportion of the cells usually resists breaking and subse- 

 quently causes heavy contamination of the nuclear fraction. Even when reagents 

 such as "hexameta" phosphate (a decalcifying material) or thioglycolic acid are used, 

 many unbroken cells are usually found. 



It has been found that if the method with very dilute citric acid at pH 6 is used 

 for isolating cell nuclei from liver, the livers must be taken from fairly young animals 

 to avoid excessive numbers of unbroken cells. This statement applies to rat, cat, 

 rabbit, and beef liver, and is probably of quite general application. As the animal 

 ages, more and more of the liver cells become resistant to breakage. It is impossible, 

 for example, to achieve sufficient breakage of cells when this method is applied to 

 cow liver. 



2. Methods Involving the Use of Nonaqueous Solvents with 

 Lyophilized Material 



a. Limitations of Methods 



The most obvious limitation to the use of nonaqueous procedures in iso- 

 lating cell nuclei is that enzyme studies are restricted by the damaging 

 action on certain enzymes of the lyophilization or of the solvents or both. 

 Studies of cytochrome oxidase, for example, should for this reason be carried 

 out on nuclei obtained in aqueous media. Mirsky et alP state that as a 

 general rule those enzymes which can be obtained from acetone powders 



