An improved method of preparing nuclei for absorption cytophotometry.
An improved method of isolating nuclei from tissue for Feulgen-DNA measurements has been developed. The optimal nuclear isolation medium (NIM) was found to be a solution of 2% polyethylene glycol (PEG) and 0.6% NP40 in phosphate-buffered saline. The disaggregation procedure consists of gentle mechanical disruption of the tissues, followed by a 10 min incubation in the NIM at room temperature. The mixture is then syringed four times through a 27-gauge needle, and the nuclei are placed onto slides with a cytocentrifuge. Nuclei prepared in NIM without PEG had obvious DNA leakage and tended to form clumps. Addition of PEG to the NIM led to separation of nuclei without any DNA leakage, thus greatly increasing the accuracy of the DNA cytophotometry results. G0/G1 nuclei at the appropriate ploidy levels were found for non-transformed and transformed tissues prepared with this technique. In addition, S-phase liver nuclei prepared in this manner showed the expected incorporation of (3H) thymidine after a 1/2 hr pulse in vivo.
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D000818:Animals; D002458:Cell Fractionation / Q000379:methods*; D002460:Cell Line; D002467:Cell Nucleus / Q000032:analysis*; D002645:Chickens; D004396:Coloring Agents; D004247:DNA / Q000032:analysis*; D003902:Detergents; D004912:Erythrocytes / Q000648:ultrastructure; D008099:Liver / Q000648:ultrastructure; D008297:Male; D051379:Mice; D009369:Neoplasms; D017830:Octoxynol; D011003:Ploidies; D011092:Polyethylene Glycols; D012394:Rosaniline Dyes; D012996:Solutions; D013194:Staining and Labeling; D013737:Testis / Q000648:ultrastructure
D004396:Coloring Agents; D003902:Detergents; C007722:Feulgen stain; D012394:Rosaniline Dyes; D012996:Solutions; D011092:Polyethylene Glycols; D017830:Octoxynol; D004247:DNA