104 ALEXANDER L. BOUNCE 



gentle methods available are used for breaking cells, whereby mitochondria 

 or nuclei are not disrupted, the erythrocytes present in the tissue will also 

 remain unbroken and will concentrate in the nuclear fractions. It is there- 

 fore necessary either to eliminate the red cells by some step which causes 

 them to become laked or else to remove them by perfusing the organ from 

 which the nuclei are to be isolated. Perfusion with isotonic sahne at to 

 5° until the organ ceases to lose color, followed by perfusion at the same 

 temperature with 0.25 M sucrose solution containing calcium chloride at 

 a concentration of 0.0018 M as advocated by Hogeboom and Schneider,^ 

 constitutes a satisfactory procedure for liver and no doubt for other organs 

 also. It is essential to use the isotonic saline before the sucrose, since the 

 latter solution alone will not wash out all the blood. In the case of liver, 

 perfusion through the portal vein is satisfactory. To avoid difficulty in 

 cannulation, the cannula should be inserted in the vein and tied before the 

 liver is removed from the animal. 



d. Description of Methods 



It would require too much space to go into the details of all methods that 

 have been published for isolating cell nuclei in aqueous media and the 

 reader is referred to the papers cited. Certain methods which have been 

 used in the writer's laboratory and which seem to be of rather general utility 

 will however be covered in some detail. 



(1) Methods Involving the Use of Citric Acid in Water Solution. The isolation of cell 

 nuclei from rat liver at pH 4.0 or slightly lower' is an example of a method which 

 can be used on a relatively large scale to obtain sufficient quantities of nuclei for the 

 subsequent isolation of nuclear constituents such as lipid and DNA, as has already 

 been stated. Homogenization is achieved either by the Waring Blendor alone or by a 

 short treatment in the Waring Blendor followed by passage through the colloid mill. 

 Water is used as suspending medium and the pH is adjusted with molar citric acid. 

 Fifty grams of frozen, chopped-up rat liver is placed in a Waring Blendor with 200 ml. 

 of ice-cold water. Seven milliliters of molar citric acid is added and the Blendor is 

 run for about 1 min. at full or slightly reduced speed. The mixture is then filtered 

 once through Curity cheesecloth grade 60, 4 layers; twice through grade 90, 4 layers; 

 and twice through grade 120, 4 layers. The pH is then checked with the Beckman pH 

 meter using a small sample of homogenate that has been removed for this purpose and 

 allowed to warm up to room temperature. In carrying out the preliminary homogeni- 

 zation, the Waring Blendor is operated in the cold room at to 3° and all filtrations 

 are also carried out in the cold room. It is desirable to add a very small amount of 

 cracked ice to the Blendor and also to add pieces of cracked ice to the portion of 

 homogenate which is filtering through the cheesecloth and to the portion in the col- 

 lecting beaker. The addition of too much ice added to the Blendor should be avoided 

 since it causes the formation of an unmanageable slush. 



The filtered preliminary homogenate can now be treated in one of two ways. It 

 can be put back into the Waring Blendor for 7 to 8 min. with the Blendor operating 

 at a reduced speed obtained by the use of a rheostat to lower the applied voltage to 



