ISOLATION OF CELL NUCLEI 455 



saline soln. by resuspending in 30-40 ml. and centrifuging each time. 

 The final suspension of nuclei has a faint yellow color, and it is 

 stable for 2 weeks when stored in a refrigerator. The nuclei may be 

 stained with aqueous methylene blue. Agglutination occurs when the 

 suspension is diluted with water but the nuclei may be suspended 

 safely in 0.1 M potassium dihydrogen phosphate soln. 



The lysolecithin is prepared by grinding the poison glands of 100 

 bees with an emulsion of 5 g. of lecithin in 20 ml. phosphate buffer 

 (pH 7.0-7.1) and allowing the mixture to digest at 37° for 24 hr. 

 The material is then filtered through a Berkfeld filter. Snake venom 

 could be substituted for the bee poison. 



NOTE. In a private communication to the writer, R. R. Bensley pointed out 

 that "it seems a pity to spend the time to pull the stings out of a hundred 

 honey bees when a few drops of ether added to a suspension of the cells 

 in salt solution will accomplish the same purpose. As a matter of fact, this 

 process of hemolysis does not isolate the nuclei since the stroma of the 

 corpuscle can be demonstrated closely contracted around the surface of 

 the nucleus." 



Bounce and Lan Procedure for Isolation of Nuclei 



Wash the red cells from fresh defibrinated blood twice with 0.9% 

 sodium chloride soln. by centrifuging and decanting. Suspend in a 

 vol. of the saline soln. equal to that of the blood used and add 

 5 ml. of 0.11 M phosphate buffer (pH 6.8-7.0), containing 0.3 g. 

 Merck's purified saponin for each 100 ml. red cell suspension. The 

 laking is complete in 5 min. Wash the nuclei liberated four to five 

 times by centrifuging, decanting and resuspending in saline soln. 

 Each time add 2-3 ml. of the 0.11 M phosphate buffer to the 

 centrifuged nuclei, without stirring, just before adding the saline 

 soln. 



The observation of Crossmon (1937), that nuclei are ejected from muscle 

 cells when a bit of the tissue is teased in 5% citric acid, led Stoneburg (1939) 

 to an application of this principle for the isolation of nuclei from beef heart 

 muscle, rabbit thigh muscle, tumor cells, and leucocytes. Bounce (1943a) has 

 pointed out that while the use of 5% citric acid may yield nuclei satisfactory 

 for studies on lipids, nucleic acids, and acid-resistant nucleoproteins, the low 

 pH involved denatures enzymes and proteins in general. Hence, Dounce (1943a) 

 modified the Stoneburg procedure to obtain nuclei suitable for enzj^me and 

 protein work. 



