PREPARATION OF ISOLATED-NUCLEI FROM K-562 HEMATOPOIETIC-CELL LINE FORHIGH-RESOLUTION SCANNING ELECTRON-MICROSCOPY

The aim of the work is to visualise nuclear pore complexes (NPCs) in m ammalian cells by high resolution scanning electron microscopy. A dete rgent-free isolation protocol was employed to obtain clean nuclei from the haemopoietic cell line K 562. Nuclear isolation was performed by mechanical homogenisation under hypotonic conditions followed by purif ication of the nuclear fraction. The isolated nuclei were attached to silicon chips, fixed, critical point dried, and sputter coated with a thin film (3-4 nm) of tantalum. Analysis of the nuclear surface by sca nning electron microscopy (SEM) revealed a strong sensitivity of the o uter nuclear membrane (ONM) to disruption during the isolation procedu re. A significant reduction of the characteristic pattern of damage to the ONM was achieved by means of an isopicnic centrifugation on an is oosmolar balanced Percoll gradient. Analysis of the population of isol ated nuclei by flow cytometry showed no signs of cell cycle specific l osses of nuclei during isolation. The SEM investigations of the morpho logy of the nuclear envelope (NE) and of substructural details of NPCs and polyribosomes were performed using an in-lens field emission scan ning electron microscope. (C) 1994 Wiley-Liss, Inc.