106 ALEXANDER L. BOUNCE 



Usually one original centrifugation and three washings are sufficient to produce 

 clean nuclei. In the last two washings, the pH of the gum may be as high as 6.2. Gum 

 ghatti adjusted to pH 6.0 can also be used instead of gum arable. ^^ It should be noted 

 however that it is not possible to use gum arable in isolating cell nuclei at pH values 

 of 4.0 or less, since in these conditions the gum forms complexes with the particulate 

 matter in the homogenates and causes massive agglutination. 



(3) Methods Involving the Use of Sucrose in Water Solution. It is possible to isolate 

 liver cell nuclei on a relatively large scale using isotonic sucrose solution as suspend- 

 ing medium, provided that the pH is lowered to 5.9 to 6.0 before homogenization in 

 the Waring Blendor or colloid mill.^"''^ Such nuclei are very similar in properties to 

 those isolated in water or gum arable solution with the pH lowered to 5.9 to 6.0. Since 

 no gel is formed on adding alkali or sodium chloride to the nuclei isolated in sucrose 

 at pH 6, it would appear that an autolytic degradation has presumably occurred. 



It is also possible to isolate liver cell nuclei in isotonic sucrose solution to which 

 calcium chloride has been added. Calcium chloride was introduced by R. Schneider 

 and Peterman'^ in isolating cell nuclei, and its use has been taken up by Hogeboom 

 et al.^'^ in the development of a small-scale method for isolating liver cell nuclei. At 

 Rochester, calcium chloride-sucrose solutions have been found very suitable in large - 

 and small-scale isolations of liver cell nuclei. A point of major interest is that calcium 

 chloride apparently inhibits autolytic changes and permits the isolation of liver cell 

 nuclei at pH values sufficiently high to allow the formation of gels with alkali or 

 saline. Concentrations of calcium chloride as low as 0.0018 M suffice under certain 

 conditions for this purpose, but higher concentrations are desirable for obtaining 

 clean nuclei. On a "macro" scale the following procedure is recommended. 



Fifty grams of frozen rat liver is mixed for about a minute in the Waring Blendor 

 with 250 ml. of 0.25 M (isotonic) sucrose solution containing calcium chloride in a con- 

 centration of 0.005 M. Filtration through cheesecloth as described previously is car- 

 ried out and the filtered preliminary homogenate is then passed twice through the 

 colloid mill at medium speed (10,000 r.p.m.) with a clearance of 1.5-thousandths of 

 an inch. The homogenate is poured into two 250-ml. centrifuge bottles and underlaid 

 with about 100 ml. of 0.0002 M CaCl2-0.34 M sucrose solution. The homogenate thus 

 obtained is centrifuged at about 2200 r.p.m. for 20 min. The supernatant is poured 

 off, care being taken not to lose too much loosely packed sediment, and the sediment 

 is then suspended with thorough shaking in about 200 ml. of 0.25 M (isotonic) plain 

 sucrose solution (without the addition of any calcium chloride). The pH is then ad- 

 justed to 6.2 withdilute citric acid (about 0.01 M), care being taken not to go below the 

 desired pH value. (A small sample of the material is warmed to room temperature 

 when testing the pH.) The suspension is then placed in two portions in two 250-ml. 

 centrifuge bottles and each portion is underlaid with about 100 ml. of 0.34 M plain 

 sucrose solution. The material is centrifuged for 30 min. at 1500 r.p.m. The supernatant 

 is discarded and the sediment is suspended in about 100 ml. of 0.25 M (isotonic) 

 sucrose solution, no pH adjustment being necessary. The material is placed in a 

 200-ml. centrifuge bottle and is underlaid with 0.34 M sucrose solution. Centrifugation 

 is carried out at 1300 r.p.m. for 30 min. 



The supernatant is poured off and the sediment is suspended in about 150 ml. of 

 1% gum arable solution which has been previously adjusted to pH 6.2. The nuclei 

 are centrifuged down for 20 min. at 1300 r.p.m. The supernatant is discarded and a 



29 E. R. M. Kay, Ph.D. Thesis, University of Rochester, 1953. 



^"K. Arnesen, Y. Goldsmith, and A. D. Dulaney, Cancer Research 9, 669 (1949). 



31 A. L. Dounce, J. Cellular Com-p. Phijsiol. 39, Suppl. 2, 43 (1952). 



