Flow cytometry of human colorectal tumors: nuclear isolation by detergent technique

A simple one-step technique for detergent isolation and DNA staining of nuclei from mouse colon and from human colorectal tumors was investigated. Nuclear yield increased with treatment time, up to 24 h. There were only minor differences when detergent concentrations from 0 to 0.6% were used. The lowest (0.03%) concentration was most effective. No loss of nuclei was effected by cell lysis and no selectivity was observed for isolation of certain cell-cycle phases or ploidy classes from heterogeneous tumors. The nuclei were stable in the stain-detergent solution for 24 h, but lymphocytes were sensitive to the possible proteolytic activity of one of the two commercial RNase preparations. Of the total number of parenchymal nuclei in mouse liver, as estimated by a stereological method, approximately 60% were isolated by the procedure (approximately 0.9 X 10(8) nuclei/g tissue). From mouse colon the average nuclear yield was 1.8 X 10(8)/g, and from human colorectal tumors 0.9 X 10(8)/g (ranges 0.3-1.9 X 10(8]. Microscopic examination of undissolved tissue fragments from the preparation of tumors and mouse colon showed a high selectivity for isolation of epithelial and neoplastic nuclei, leaving the stroma with its nuclei almost intact.