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dc.contributor.authorReipert, Siegfried
dc.contributor.authorReipert, Brigit M
dc.contributor.authorAllen, Terence D
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.
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
html.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.


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