Microcarriers for high-pressure freezing and cryosectioning of adherent cells.
A method is described employing microcarrier spheres of cross-linked dextran for obtaining ultra- and semithin vitreous sections from high-pressure frozen anchorage-dependent (mammalian) cells. Avoiding trypsination or scraping cells off from the culture surface, the presented approach allows for cryoimmobilization, cryosectioning and cryoelectron microscopy/tomography of frozen-hydrated cells in an unperturbed manner which is important to preserve the native state of, for instance, the cytoskeleton. Furthermore, our studies on the 'life cycle' of Herpes simplex virus in Vero cells demonstrate that cell monolayers on microcarrier beads are well suited for fluorescence microscopic characterization of the sample prior to high-pressure freezing.
Hagen
C
C
Department of Molecular Structural Biology, Max Planck Institute of Biochemistry, D-82152 Martinsried, Germany. hagen@biochem.mpg.de
Grünewald
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K
D000818:Animals; D002448:Cell Adhesion; D018929:Cell Culture Techniques; D002522:Chlorocebus aethiops; D020285:Cryoelectron Microscopy / Q000379:methods*; D015925:Cryopreservation; D016616:Cryoultramicrotomy / Q000295:instrumentation*; D003911:Dextrans; D005615:Freezing; D005629:Frozen Sections; D018259:Herpesvirus 1, Human / Q000502:physiology / Q000648:ultrastructure; D008856:Microscopy, Fluorescence / Q000379:methods*; D008863:Microspheres; D014709:Vero Cells / Q000648:ultrastructure / Q000821:virology
D003911:Dextrans; C052839:Cytodex