///. ISOLATION OF CELL NUCLEI 



The isolation of cell nuclei for purposes of chemical investiga- 

 tion seems to have been attempted first by Miescher (1871), who 

 digested leucocytes with pepsin to remove the cytoplasm; the 

 nuclei could then be collected on a filter. Other early attempts at 

 the separation of nuclei were made by Ackermann ( 1904 ) , who 

 laked chicken erythrocytes in distilled water and precipitated the 

 nuclei in 3.6% sodium chloride solution, and Warburg (1910), who 

 employed a freezing-thawing technique to disrupt the reel cells. 



Since a number of methods for the isolation of nuclei have been 

 evolved during the past 15 years, each with its particular advantages 

 and drawbacks, and each more suited to certain types of cells than 

 to others, the most important of these methods will be described in 

 detail so that the investigator may use his own judgment in their 

 application. 



Since the Warburg (1910) method of freezing and thawing results 

 in partial agglutination and damage to erythrocytes, and incom- 

 pletely hemolyzes them, Laskowski (1942) developed a technique 

 based on hemolysis by lysolecithin. In some instances, as in the case 

 of lipid studies, lysolecithin treatment would be undesirable; hence 

 Bounce and Lan (1943) employed saponin for the hemolysis. 



A. NUCLEI FROM AVIAN ERYTHROCYTES 



Laskowski Procedure for Isolation of Nuclei 



Centrifuge the erythrocytes from 30—40 ml. citrated blood, and 

 wash them several times with isotonic saline soln. by successive 

 centrifugations and decantations. After the last centrifugation, 

 pipette out the red cells from the bottom of the layer and suspend 

 them in 30-40 ml. saline soln. Add 5-8 ml. lysolecithin soln. and 

 determine when hemolysis is complete by microscopic examination; 

 it usually takes 20-40 min. at room temperature. Centrifuge the 

 liquid, and wash the material thrown down five to six times with 



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