Preparation of isolated nuclei from K 562 haemopoietic cell line for high resolution scanning electron microscopy.
Affiliation
CRC Department of Structural Cell Biology, Paterson Institute for Cancer Research, Christie Hospital NHS Trust, Manchester, United Kingdom.Issue Date
1994-09-01
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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.Citation
Preparation of isolated nuclei from K 562 haemopoietic cell line for high resolution scanning electron microscopy. 1994, 29 (1):54-61 Microsc. Res. Tech.Journal
Microscopy Research and TechniqueDOI
10.1002/jemt.1070290108PubMed ID
8000085Type
ArticleLanguage
enISSN
1059-910Xae974a485f413a2113503eed53cd6c53
10.1002/jemt.1070290108
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