456 MECHANICAL SEPARATION OF CELLULAR COMPONENTS 



B. NUCLEI FROM OTHER CELLS 



Stoneburg Procedure for Isolation of Nuclei 



Nuclei from Muscle Tissue. Grind the tissue, cleaned of visible 

 fat, as fine as possible in a meat chopper. Place 100 g. in a 1 1. beaker 

 and cover with 500 ml. of 5% citric acid. Stir occasionally and let 

 stand overnight. Skim off sm'face fat, dilute soln. with an equal vol. 

 water, and filter through eight layers of cheesecloth. Run filtrate 

 through a Sharpies centrifuge at 26,000 R.P.M. The centrifuge 

 cylinder is lined with cellophane to prevent the solvent action of 

 the acid on the metal. The material washed off the cellophane sheet 

 will contain about 50% nuclei. Wash the material four times by 

 successive centrifugations and resuspensions in water. The nuclei 

 content will be raised to about 70% by this process. Digest the 

 residue in the centrifuge bottle with 250 ml. 1% hydrochloric acid 

 and 250 ml. 0.8% sodium chloride soln. containing 2-4 g. pepsin 

 (strength 1:10,000) for 4 hr. at 37°. Stir occasionally, and at the 

 end siphon off the supernatant from the settled nuclei. Wash the 

 nuclei with distilled water and then centrifuge. A smear of the 

 residue should show nuclei undamaged histologically and free from 

 debris. 



Nuclei from Rat Tumor Tissue. After removal of necrotic 

 material the tissue is ground in a meat chopper, treated with citric 

 acid soln., and filtered as in the case of muscle. Add 1 vol. water to 

 the filtrate and let stand 3 hr. Omit supercentrifugation but other- 

 wise continue the procedure given for muscle. 



Nuclei from Leucocytes. Add pus to five times its vol. of 5% 

 citric acid. The nuclei settle rapidly and are then subjected to the 

 procedure given for tumor tissue. 



Dounce Procedure for the Isolation of Nuclei 



Nuclei from Rat Liver. Make up approximately equal vol. 

 cracked ice and water to 500 ml. and add 1.05 ml. 1 M citric acid 

 (final cone. M/475, pH 6.0-6.2). Place the mixture in a Waring 

 blendor and add 100 g. frozen rat liver as rapidly as possible without 

 stalling the stirrer. The liver may be frozen conveniently in the freez- 



