Isolation of nuclei in media containing an inert polymer to mimic the crowded cytoplasm.

Methods in molecular biology (Clifton, N.J.)

PubMedID: 25311119

Hancock R, Hadj-Sahraoui Y. Isolation of nuclei in media containing an inert polymer to mimic the crowded cytoplasm. Methods Mol Biol. 2014;122825-32.
Within cells, the nucleus is surrounded by the cytoplasm which contains diffusible macromolecules at a high concentration (>100 mg/ml). When cells are broken to isolate nuclei by current methods these macromolecules are dispersed, and to reproduce the environment of nuclei in vivo more closely we have developed a method to isolate them in a medium where cytoplasmic macromolecules are replaced by an inert, volume-occupying polymer and which is essentially cation-free. Nuclei isolated by this method resemble closely those prepared by conventional procedures as seen by optical and electron microscopy, and their internal compartments (nucleoli, PML and Cajal bodies, transcription centers, and splicing speckles) and transcriptional activity are conserved. This procedure is efficient for mammalian cells that normally grow in suspension and do not have an extensive cytoskeleton, and requires ~30 min.