458 MECHANICAL SEPARATION OF CELLULAR COMPONENTS 



This may be prevented by adding a few drops of 1 M citric acid to 

 tlie wash water each time. Actually less washing is required at pH 

 3.8 to 4.0 than at 6.0 to 6.2 since the nuclei pack better on centrifug- 

 ing. 



Nuclei from Tumor Tissue. In a later paper Dounce ( 1943c) 

 described the application of the preceding method to tumor cells. In 

 the case of Walker carcinosarcoma 256, add 37.6 ml. 1 M citric acid 

 in the blendor to the 500 ml. of mixture containing 100 g. tissue. Run 

 the blendor for 15 min. ; the final pH is 3.0. After the first washing, 

 add a drop or more of 1 M citric acid to prevent agglutination. The 

 reason for the lower pH in this instance is that cytoplasm cannot be 

 removed very well from the nuclei at pH 3.8 to 4.0. When hepatoma 

 31 was used, it was impossible to obtain a good preparation in the 

 pH range 3.0 to 4.0 necessitating the addition or 100 ml. 1 M citric 

 acid to the 500 ml. vol. The final pH was 2.4. Here, too, citric acid is 

 added in the washings to prevent agglutination. 



The Dounce pioceduie has been criticized by Hoerr (1943), who claims 

 that 0.85% sodium chloride is preferable to distilled water for extractions 

 and washings, and who objects to the use of the Waring blendor as being 

 too drastic a treatment leading to a certain degree of gelling. While Hoerr 

 may be right, and even though it is obvious that the less drastic the treatment 

 of biological systems to attain a given end, the better, the fact that Dounce 

 obtained good preparations which were suitable for a variety of studies on 

 enzymes and other nuclear constituents cannot be disregarded. Hoerr favors 

 the Lazarow (1941, 1943) procedure of breaking up cells by forcing a suspension 

 of them through bolting silk, and handling the material at a temperature 

 just above 0° taking care not to let it freeze. 



Lazarow Procedure for Isolation of Liver Nuclei 

 as Used by Hoerr 



Perfuse the liver in situ with cold physiological salt soln. by re- 

 peated variations of hydrostatic pressure from 2 to 4 ft., clamping the 

 inferior vena cava for 10-20 sec. each min., and massaging the liver 

 through the abdominal wall while the blood is flowing freely from 

 the vein. Usually 500-700 ml. saline soln. is required to remove prac- 

 tically all the blood cells. Remove the liver and chill to 0° as rapidly 

 as possible, but do not allow to freeze. Triturate the tissue briefly 

 in a mortar with 0.85% sodium chloride soln. and gently knead 

 through bolting silk. Suspend the emulsion in 0.5 to 0.7% sodium 

 chloride soln. at a pH of 6.0 to 6.2. Separate the nuclei from por- 



