ISOLATION OF CELL NUCLEI 457 



ing compartment of a refrigerator and it should be used as soon as 

 frozen. Run the blendor until all the ice has melted (10-15 min.). 

 Strain the mixture through two layers of fine cheesecloth (twenty 

 threads per cm.) and, when the cloth becomes clogged, replace it 

 with new material. Wring out the cloths used and strain the liquid 

 into the already strained batch. Repeat the straining with four 

 layers of cheesecloth and centrifuge for 20 min. at 1500-2000 R.P.]\I. 

 in 250 ml. centrifuge bottles. Decant down to the first line demark- 

 ing the supernatant and the more loosely packed sediment. Add 

 distilled water to the sediment to make a final vol. of 400 ml. 

 and stir well to break up lumps. A drop of capryhc alcohol may 

 be used to break the foam. Centrifuge the suspension for 15 

 min. and again discard the supernatant fluid. Stir the residue with 

 about 400 ml. distilled water and centrifuge for 10 min. Wash 

 sediment with 200 ml. distilled water and centrifuge 5 min. at a 

 slower speed (1000-1500 R.P.M.). Discard the supernatant and stir 

 the nuclei with 200 ml. distilled water. Centrifuge for only 3 min. 

 this time and at a still slower speed. Repeat the washing twice with 

 200 ml. portions of distilled water and centrifuge 3 min. each time 

 at the lower speed. Before the last centrifugation, pass the suspension 

 through four layers of cheesecloth. Stir the nuclei well with about 100 

 ml. distilled water and let stand for 45 min. in a 100 ml. cylinder. 

 Carefully decant the top 95 ml. containing most of the nuclei from 

 the whole cells on the bottom. Recover the nuclei by centrifuging and 

 resuspending in a small vol. distilled water. The light reddish-brown 

 color of the final preparation is due to adsorbed hemoglobin. 



There are some additional points that should be mentioned. At a 

 pH 6.5, or higher, the cells rupture in the presence of distilled water 

 but the purified nuclei do not seem to be harmed. In the pH range of 

 4.0 to 5.9 cytoplasmic granules agglutinate to a solid mass on cen- 

 trifugation making it impossible to separate nuclei, while at 3.8 to 

 4.0 good nuclei preparations are obtained but the acidity results in 

 some alteration of enzymes and proteins. Adsorbed hemoglobin can 

 be removed from the product to some extent by one washing, and 

 completely by two, with Ringer soln. at pH 7.4. However, the Ringer 

 soln. extracts a certain amount of protein from the nuclei and causes 

 them to shrink. Subsequently, Dounce (1943b) showed that in the 

 preparation of nuclei at pH 3.8 to 4.0 the pH tends to rise above 4.0 

 on successive washings with the result that the nuclei agglutinate. 



