A rapid method for the isolation of nuclei from Ehrlich ascites tumor cells.

Abstract

Summary A method for the isolation of nuclei from Ehrlich ascites tumor cells free of cytoplasmic contamination is described. The procedure involves hypotonic osmotic shock, homogenization with a close-fitting Dounce homogenizer, and differential centrifugation through two different high-density sucrose solutions. The extent of cytoplasmic contamination of the isolated nuclei at each stage was followed by DNA, RNA, protein, and marker enzyme analysis. The final product was examined by phase and electron microscopy and indicated the presence of a high yield of morphologically intact Ehrlich ascites tumor cell nuclei, free of cytoplasmic tags. Isolated ascites cell nuclei contained nicotinamide adenine dinucleotide glycohydrolase (EC 3.2.2.5) which could not be solubilized by lipase treatment and which differed significantly from the microsomal enzyme in its pH optimum and heat stability.