The Production of Culture: Media and the Urban Arts

The ultrastructure of cells suspended in physiological fluids or cell culture media can only be studied if the living processes are stopped while the cells remain in suspension. Attachment of living cells to carrier surfaces to facilitate further processing for electron microscopy produces a rapid reorganization of cell structure eradicating most traces of the structures present when the cells were in suspension. The structure of cells in suspension can be immobilized by either chemical fixation or, much faster, by rapid freezing (cryo-immobilization). The fixation speed is particularly important in studies of cell surface reorganization over time. High pressure freezing provides conditions where specimens up to 500μm thick can be frozen in milliseconds without ice crystal damage. This volume is sufficient for cells to remain in suspension until frozen. However, special procedures are needed to assure that the unattached cells are not lost during subsequent processing for LVSEM or HVEM using freeze-substitution or freeze drying. We recently developed such a procedure.

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Kalinin: A new epithelial basement membrane adhesion molecule

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s0424820100085198 ◽

1991 ◽

Vol 49 ◽

pp. 178-179

Author(s):

Douglas R. Keene ◽

Gregory P. Lunstrum ◽

Patricia Rousselle ◽

Robert E. Burgeson

Keyword(s):

Basement Membrane ◽

Culture Media ◽

Polyacrylamide Gel Electrophoresis ◽

Human Keratinocytes ◽

Positive Staining ◽

Human Amnion ◽

Epithelial Basement Membrane ◽

Type Vii Collagen ◽

Keratinocyte Cell ◽

Epithelial Basement

A mouse monoclonal antibody produced from collagenase digests of human amnion was used by LM and TEM to study the distribution and ultrastructural features of an antigen present in epithelial tissues and in cultured human keratinocytes, and by immunoaffinity chromatography to partially purify the antigen from keratinocyte cell culture media.By immunofluorescence microscopy, the antigen displays a tissue distribution similar to type VII collagen; positive staining of the epithelial basement membrane is seen in skin, oral mucosa, trachea, esophagus, cornea, amnion and lung. Images from rotary shadowed preparations isolated by affinity chromatography demonstrate a population of rod-like molecules 107 nm in length, having pronounced globular domains at each end. Polyacrylamide gel electrophoresis suggests that the size of this molecule is approximately 440kDa, and that it is composed of three nonidentical chains disulfide bonded together.

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