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Preparation of isolated nuclei from K 562 haemopoietic cell line for high resolution scanning electron microscopy
Author(s) -
Reipert Siegfried,
Allen Terence David,
Reipert Birgit Maria
Publication year - 1994
Publication title -
microscopy research and technique
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.536
H-Index - 118
eISSN - 1097-0029
pISSN - 1059-910X
DOI - 10.1002/jemt.1070290108
Subject(s) - scanning electron microscope , electron microscope , nuclear pore , microscopy , biophysics , materials science , analytical chemistry (journal) , percoll , chemistry , biology , centrifugation , chromatography , cytoplasm , optics , biochemistry , physics , composite material
The aim of the work is to visualise nuclear pore complexes (NPCs) in mammalian cells by high resolution scanning electron microscopy. A detergent‐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 purification 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 scanning electron microscopy (SEM) revealed a strong sensitivity of the outer nuclear membrane (ONM) to disruption during the isolation procedure. A significant reduction of the characteristic pattern of damage to the ONM was achieved by means of an isopicnic centrifugation on an isoosmolar balanced Percoll gradient. Analysis of the population of isolated nuclei by flow cytometry showed no signs of cell cycle specific losses of nuclei during isolation. The SEM investigations of the morphology of the nuclear envelope (NE) and of substructural details of NPCs and polyribosomes were performed using an in‐lens field emission scanning electron microscope. © 1994 Wiley‐Liss, Inc.