Abstract
The early attempts to separate the nuclei from avian erythrocytes resulted in obtaining an amorphous mass of nuclear material. Warburg 1 was the first to isolate the separate nuclei. He used a freezingmelting technic. The same method was used by Miyake. 2 The disadvantages of this method are: partial agglutination and damage of nuclei, incomplete hemolysis and the necessity of further separation.
The method to be described leads to a stable suspension of free nuclei which probably have been less damaged than in the freezing technic. The method is based on the hemolysis of hen erythrocytes with lysolecithin in neutral saline solution, washing the nuclei with saline and resuspension in saline.
Lysolecithin was prepared as follows: Lecithin emulsion (0.5 g per 20 ml) was made in phosphate buffer pH 7.0-7.1 (crude lecithin recovered from cadmium salt of student preparation was used). The lecithin emulsion was ground with poison glands of 100 bees (lecithinase A), incubated during 24 hours at 37°C and filtered through a Berkefeld filter.
Chicken blood was obtained by cardiac puncture, according to the method of Sloan and Wilgus. 3 Citrate was used to prevent clotting. The blood was centrifuged at a speed just sufficient to sediment the erythrocytes and leave the bulk of the leucocytes in suspension. The plasma with suspended leucocytes and the top layer of erythrocytes were pipetted off. Isotonic saline solution was added to the rest of the erythrocytes and the procedure was repeated several times (5-6). Finally, the erythrocytes were centrifuged to close packing, the pipette introduced to the bottom of the centrifuge tube, and the lower layer of erythrocytes pipetted into a second centrifuge tube and suspended in saline.
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