Detergent free fractionation of nuclei from mouse embryonic stem cells

Mouse embryonic stem cells (ESCs), with their ability to self-renewal, and to differentiate into virtually all types, have become the major focus in contemporary cell-based therapy and regenerative medicine. Many studies about the signaling mechanism regulating self-renewal or differentiation have been performed in mouse ESCs. Pure nuclei fractionation is needed for the studies of gene regulation involved in self-renewal or differentiation of ESCs. Here we develop more efficient method for isolation of pure nuclei from mouse ESCs than the conventional method. We isolated more pure nuclei fraction with detergent-free buffer by freezing-thawing cycling. Mouse ESC nuclei isolated by our method were more pure than those by the conventional method. Oct4, a well-known stemness marker and nucleus protein, in the nuclei fraction, specifically bound to the oct4 binding site. Light electroscopy and electron microscopy showed the intactness of the isolated nuclei fraction. Flow cytometric analysis of the population of isolated nuclei revealed no signs of cell cycle specific losses of nuclei during isolation procedure. This contributes to studying nuclear signaling molecules involved in self-renewal and differentiation in mouse ESCs.