Isolation of chromosome clusters from metaphase-arrested HeLa cells.
We have developed a simplified approach for the isolation of metaphase chromosomes from HeLa cells. In this method, all the chromosome from a cell remain together in a bundle which we call a "metaphase chromosome cluster". Cells are arrested to 90-95% in metaphase, collected by centrifugation, extracted with non-ionic detergent in a low ionic strength buffer at neutral pH, and homogenised to strip away the cytoskeleton. The chromosome clusters which are released can then be isolated in a crude state by pelleting or they can be purified away from nearly all the interphase nuclei and cytoplasmic debris by banding in a PercollTM density gradient. -- This procedure has the advantages that it is quick and easy, metaphase chromatin is recovered in high yield, and Ca++ is not needed to stabilise the chromosomes. Although the method does not yield individual chromosomes, it is nevertheless very useful for both structural and biochemical studies of mitotic chromatin. The chromosome clusters also make possible biochemical and structural studies of what holds the different chromosomes together. Such information could be useful in improving chromosome isolation procedures and for understanding suprachromosomal organisation of the nucleus.
D002458:Cell Fractionation; D002499:Centrifugation, Density Gradient; D002877:Chromosomes, Human / Q000502:physiology* / Q000648:ultrastructure; D006367:HeLa Cells / Q000502:physiology; D006801:Humans; D008677:Metaphase