Preparation of isolated nuclei from K 562 haemopoietic cell line for high resolution scanning electron microscopy.

2.50
Hdl Handle:
http://hdl.handle.net/10541/96086
Title:
Preparation of isolated nuclei from K 562 haemopoietic cell line for high resolution scanning electron microscopy.
Authors:
Reipert, Siegfried; Reipert, Brigit M; Allen, Terence D
Abstract:
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.
Affiliation:
CRC Department of Structural Cell Biology, Paterson Institute for Cancer Research, Christie Hospital NHS Trust, Manchester, United Kingdom.
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 Technique
Issue Date:
1-Sep-1994
URI:
http://hdl.handle.net/10541/96086
DOI:
10.1002/jemt.1070290108
PubMed ID:
8000085
Type:
Article
Language:
en
ISSN:
1059-910X
Appears in Collections:
All Paterson Institute for Cancer Research

Full metadata record

DC FieldValue Language
dc.contributor.authorReipert, Siegfrieden
dc.contributor.authorReipert, Brigit Men
dc.contributor.authorAllen, Terence Den
dc.date.accessioned2010-04-09T10:19:36Z-
dc.date.available2010-04-09T10:19:36Z-
dc.date.issued1994-09-01-
dc.identifier.citationPreparation of isolated nuclei from K 562 haemopoietic cell line for high resolution scanning electron microscopy. 1994, 29 (1):54-61 Microsc. Res. Tech.en
dc.identifier.issn1059-910X-
dc.identifier.pmid8000085-
dc.identifier.doi10.1002/jemt.1070290108-
dc.identifier.urihttp://hdl.handle.net/10541/96086-
dc.description.abstractThe 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.en
dc.language.isoenen
dc.subjectCultured Tumour Cellsen
dc.subjectAcute Erythroblastic Leukaemiaen
dc.subject.meshCell Nucleus-
dc.subject.meshCell Separation-
dc.subject.meshFlow Cytometry-
dc.subject.meshHumans-
dc.subject.meshLeukemia, Erythroblastic, Acute-
dc.subject.meshMicroscopy, Electron, Scanning-
dc.subject.meshNuclear Envelope-
dc.subject.meshTumor Cells, Cultured-
dc.titlePreparation of isolated nuclei from K 562 haemopoietic cell line for high resolution scanning electron microscopy.en
dc.typeArticleen
dc.contributor.departmentCRC Department of Structural Cell Biology, Paterson Institute for Cancer Research, Christie Hospital NHS Trust, Manchester, United Kingdom.en
dc.identifier.journalMicroscopy Research and Techniqueen
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