Nuclei from Normal and Leukemic Mouse Spleen

In the course of a study of the pentosenucleic acid (PNA) distribution in normal and leukemic mouse spleen, it was found desirable to determine the nucleic acid content of isolated nuclei. The pro cedures commonly employed for the isolation of nuclei (1, 4, 8, 13) involve the use of citric acid. This was felt to be unsuitable for the isolation of nuclei free of cytoplasmic PNA, since it has been shown (92) that the cytoplasmic nucleoproteins flocculate below p11 6.0 and precipitate at pH 4.8 or lower. The danger of contamination is particu larly great in leukemic spleen, where the cytoplas mic PNA concentration is extremely high (9). Al though the preparation of nuclei at pH 6.0 (4) might have been feasible, a procedure which avoided the use of acid entirely seemed more desirable. When attempts were made to isolate nuclei in the 0.88 M sucrose medium introduced by Hoge boom, Schneider, and Pallade (6) for the isolation of mitochondnia, the nuclei were clumped, dis torted, and badly contaminated with debris. A search was therefore made for some ion which would “harden― the nuclei without causing agglu tination of cytoplasm. Of the various salts tried, those containing divalent cations appeared most satisfactory ; equally good results were obtained with calcium chloride and manganese sulfate. In the procedure described below, 0.0018 M calcium chloride in 0.88 M sucrose was used. The method is simple, convenient and rapid, being complete with in 1 hour of the death of the animals.