Antigenic properties of nuclei segregated from spleens of normal and leukemic mice.

Various procedures have been described for the segregation of nuclei from mammalian tissues (1, 92,3, 4, 5, 6). These methods are based on a mechanical breakdown of the cells suspended in aqueous solutions of citric acid, with subsequent separation of the nuclei by centrifugation at dif ferent speeds. In our work with mouse spleens we have inves tigated other reagents but so far, we have not been able to obtain isolated nuclei without the addition of citric acid. Physiological saline solution is un satisfactory since the unbroken cells, nuclei, and cytoplasmic granules agglutinate making it im possible to separate these fractions by differential centnifugation. Potassium chloride, of the same molar concentration as the sodium chloride, like wise proved unsuitable. Various concentrations of sucrose have been used. We have found that nucleoprotein is cx tracted from the nuclei in both 8.5 per cent (iso tonic) and 30 per cent sucrose solutions. When the tissue suspensions were homogenized initially in the sucrose solutions and centrifuged, the sedi ment was found to be held in a gel which could be dissolved in molar sodium chloride solution. Upon subsequent centnifugation and dilution of the supernate to constitute a 0.14 molar NaCl solu tion, a fibrous precipitate formed. This stained with methyl green and was therefore presumed to consist of desoxyribose nucleoprotein (7). The ad dition of very small amounts of citric acid to the sucrose solution prevented the extraction of nu cleoprotein, as judged by the above criterion. Relatively more citric acid was required for the isolation of nuclei from the leukemic than from the normal tissues. A concentration of M/150 to M/1925