A reliable method for obtaining matched replicas of freeze-fractured cell suspensions

1980 ◽  
Vol 42 (1) ◽  
pp. 389-400
Author(s):  
M.H. Wilkinson ◽  
D.H. Northcote
Keyword(s):  
Reliable Method ◽  
Freeze Fracture ◽  
Cell Suspensions ◽  
Animal Cells ◽  
Fracture Characteristics ◽  
Sample Digestion ◽  
Isolated Protoplasts ◽  
Glass Capillaries ◽  
Excellent Preservation ◽  
Membrane Ultrastructure

A method that overcomes the majority of practical problems involved in performing matched freeze-fracture replication of cell suspensions is described. Specimens are rapidly frozen between copper support plates. The prior attachment of gold grids to the plates using Formvar ensures the exact alignment of corresponding grid squares and the production of a central fissure running through the specimen upon fracturing. Grids, with replicas permanently attached, are removed from the support plates by dissolving the Formvar. Sample digestion and rinsing of replicas is by a gentle procedure employing glass capillaries as a means of handling the grids. Matched replication of plant and animal cells has been achieved by this methods. Apart from the excellent preservation of the membrane ultrastructure of unfixed cells and the capacity for matching both membrane halves this technique also enhances the fracture characteristics of certain specimens, notably isolated protoplasts, providing larger fracture faces than are obtained by the knife-splintering method.

Plasma membrane ultrastructure during plant protoplast plasmolysis, isolation and wall regeneration: a freeze-fracture study

1980 ◽  
Vol 42 (1) ◽  
pp. 401-415
Author(s):  
M.J. Wilkinson ◽  
D.H. Northcote
Keyword(s):  
Plasma Membrane ◽  
Time Course ◽  
Freeze Fracture ◽  
Fracture Morphology ◽  
Plant Protoplast ◽  
Suspension Cells ◽  
Isolated Protoplasts ◽  
Fracture Study ◽  
Membrane Ultrastructure ◽  
Hexagonal Arrays

The freeze-fracture morphology of the plasma membrane of cells and isolated protoplasts of plant callus suspensions has been investigated. Plasmolysis of suspension cells leads to the formation of 2 types of hexagonal arrays of intramembrane particles situated on the inner fracture face (PF). These arrays are interpreted as proteins that have ‘crystallized’ in the plane of the membrane as the area of surrounding lipid bilayer is reduced during protoplast retraction from the cell wall. Time-course studies have revealed no positive relationship between the distribution of hexagonal arrays and the occurrence of microfibrils regenerated around isolated protoplasts during periods of culture. No evidence for the specialized transport functions attributed to hexagonal arrays of plant cells by previous workers has been found.

Transient correction of genetic defects in cultured animal cells by introduction of functional proteins

1987 ◽  
Vol 7 (8) ◽  
pp. 3012-3017
Author(s):  
D Ortiz ◽  
M M Baldwin ◽  
J J Lucas
Keyword(s):  
Genetic Defects ◽  
Wild Type ◽  
Animal Cells ◽  
Culture Dish ◽  
Tissue Culture Dish ◽  
Cell Extracts ◽  
Glass Capillaries ◽  
Hypoxanthine Guanine Phosphoribosyltransferase ◽  
Scrape Loading ◽  
Plastic Tissue Culture

Material was introduced into cultures of cells by using the method of scrape loading, in which cells are simply rubbed from the surface of a plastic tissue culture dish by a rubber-tipped rod in the presence of a macromolecule of interest. The volume of solution introduced into cells was comparable to that generally injected in the direct microinjection method with glass capillaries, that is, about 50 to 100 fl per cell. Genetic defects (lack of hypoxanthine-guanine phosphoribosyltransferase and thymidine kinase) in several cell lines were transiently corrected by scraping the cells in the presence of crude cell extracts prepared from wild-type cells.

scholarly journals Transient correction of genetic defects in cultured animal cells by introduction of functional proteins.

1987 ◽  
Vol 7 (8) ◽  
pp. 3012-3017 ◽  
Author(s):  
D Ortiz ◽  
M M Baldwin ◽  
J J Lucas
Keyword(s):  
Genetic Defects ◽  
Wild Type ◽  
Animal Cells ◽  
Culture Dish ◽  
Tissue Culture Dish ◽  
Cell Extracts ◽  
Glass Capillaries ◽  
Hypoxanthine Guanine Phosphoribosyltransferase ◽  
Scrape Loading ◽  
Plastic Tissue Culture

Material was introduced into cultures of cells by using the method of scrape loading, in which cells are simply rubbed from the surface of a plastic tissue culture dish by a rubber-tipped rod in the presence of a macromolecule of interest. The volume of solution introduced into cells was comparable to that generally injected in the direct microinjection method with glass capillaries, that is, about 50 to 100 fl per cell. Genetic defects (lack of hypoxanthine-guanine phosphoribosyltransferase and thymidine kinase) in several cell lines were transiently corrected by scraping the cells in the presence of crude cell extracts prepared from wild-type cells.

scholarly journals Fura-2 measurement of cytosolic free Ca2+ in monolayers and suspensions of various types of animal cells.

1987 ◽  
Vol 105 (5) ◽  
pp. 2145-2155 ◽  
Author(s):  
A Malgaroli ◽  
D Milani ◽  
J Meldolesi ◽  
T Pozzan
Keyword(s):  
Human Skin ◽  
Single Cells ◽  
Calibration Procedure ◽  
Cell Suspensions ◽  
Skin Fibroblasts ◽  
Homogeneous Distribution ◽  
Human Skin Fibroblasts ◽  
Animal Cells ◽  
Extracellular Medium ◽  
Fura 2

The fluorescent indicator fura-2 has been applied to a variety of cell types in order to set up appropriate conditions for measurements of the cytosolic concentration of free ionized Ca2+ [( Ca2+]i) in both cell suspensions and single cells analyzed in a conventional fluorimeter or in a fluorescence microscope equipped for quantitative analyses (with or without computerized image analyses), respectively. When the usual procedure for fluorescence dye loading (i.e., incubation at 37 degrees C with fura-2 acetoxy-methyl ester) was used, cells often exhibited a nonhomogeneous distribution of the dye, with marked concentration in multiple small spots located preferentially in the perinuclear area. These spots (studied in detail in human skin fibroblasts), were much more frequent in attached than in suspended cells, and were due to the accumulation (most probably by endocytosis) of the dye within acidic organelles after hydrolysis by lysosomal enzyme(s). When loading with fura-2 was performed at low (15 degrees C) temperature, no spots appeared, and cells remained diffusely labeled even after subsequent incubation at 32-37 degrees C for up to 2 h. Homogeneous distribution of the dye is a prerequisite for appropriate [Ca2+]i measurement. In fact, comparison of the results obtained in human skin fibroblasts labeled at either 37 or 15 degrees C demonstrated in spotty cells a marked apparent blunting of Ca2+ transients evoked by application of bradykinin. Additional problems were encountered when using fura-2. Leakage of the dye from loaded cells to the extracellular medium markedly affected the measurements in cell suspensions. This phenomenon was found to depend on the cell type, and to markedly decrease when temperature was lowered, suggesting the involvement of a facilitated transport. Calibration of fluorescence signals in terms of absolute [Ca2+]i was complicated by the increased fluorescence of fura-2 in the intracellular environment. To solve this problem we propose an in situ calibration procedure based on measurements carried out on cells in which [Ca2+]i was massively lowered (by loading the probe in a Ca2+-free medium) or increased (by treatment with the Ca2+ ionophore ionomycin, applied in a medium containing 3 mM Ca2+). These results provide explanations and, at least partial, solutions to the major problems encountered when using fura-2, and should thus be of help in clarifying the proper usage of the dye in [Ca2+]i measurements.

Freeze-fracture characteristics of insect gustatory and olfactory sensilla. II. Cuticular features

1984 ◽  
Vol 179 (3) ◽  
pp. 305-321 ◽  
Author(s):  
Fran M. van der Wolk ◽  
B. Ph. M. Menco ◽  
H. Van Der Starre
Keyword(s):  
Freeze Fracture ◽  
Olfactory Sensilla ◽  
Fracture Characteristics
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