Separation of cytoplasts and whole cells using density gradients of renografin

Abstract

Cytoplasts prepared from L929 or Hepa-2 cells were separated from whole cells using density gradients of renografin. Using this technique, cytoplasts can be isolated from cell lines which cannot be routinely enucleated with an efficiency of 100%. The purified cytoplasts excluded the vital dye trypan blue and were utilized in nuclear transplantation experiments to reconstruct whole viable cells capable of division. In addition, the renografin gradient technique was used to separate the newly reconstructed cells from any contaminating "non-renucleated" cytoplasts. This will permit immediate biochemical characterization of cytoplasmic-nuclear hybrid cells without interference from contaminating cytoplasts.