scholarly journals Calibration of the single cell gel electrophoresis assay, flow cytometry analysis and forward mutation in Chinese hamster ovary cells

Mutagenesis ◽  
1998 ◽  
Vol 13 (1) ◽  
pp. 81-84 ◽  
Author(s):  
E. D. Wagner ◽  
A.L. Rayburn ◽  
D. Anderson ◽  
M. J. Plewa
Keyword(s):  
Flow Cytometry ◽  
Single Cell ◽  
Chinese Hamster Ovary ◽  
Gel Electrophoresis ◽  
Chinese Hamster Ovary Cells ◽  
Chinese Hamster ◽  
Flow Cytometry Analysis ◽  
Ovary Cells ◽  
Single Cell Gel Electrophoresis ◽  
Forward Mutation

Efficient enrichment of high-producing recombinant Chinese hamster ovary cells for monoclonal antibody by flow cytometry

2015 ◽  
Vol 120 (3) ◽  
pp. 340-346 ◽  
Author(s):  
Takeshi Okumura ◽  
Kenji Masuda ◽  
Kazuhiko Watanabe ◽  
Kenji Miyadai ◽  
Koichi Nonaka ◽  
...  
Keyword(s):  
Monoclonal Antibody ◽  
Flow Cytometry ◽  
Chinese Hamster Ovary ◽  
Chinese Hamster Ovary Cells ◽  
Chinese Hamster ◽  
Ovary Cells

scholarly journals Selection of mutant Chinese hamster ovary cells altered glycoproteins by means of tritiated fucose suicide.

1981 ◽  
Vol 1 (10) ◽  
pp. 902-909 ◽  
Author(s):  
C B Hirschberg ◽  
R M Baker ◽  
M Perez ◽  
L A Spencer ◽  
D Watson
Keyword(s):  
Chinese Hamster Ovary ◽  
Gel Electrophoresis ◽  
Trichloroacetic Acid ◽  
Chinese Hamster Ovary Cells ◽  
Sodium Dodecyl ◽  
Chinese Hamster ◽  
Wild Type ◽  
Ovary Cells ◽  
Replica Plating ◽  
Selection Of

Mutant Chinese hamster ovary cells altered in glycoproteins have been isolated by selecting for ability to survive exposure to [6-3H]fucose. Mutagenized wild-type cells were permitted to incorporate [3H]fucose to approximately 1 cpm of trichloroacetic acid-insoluble radioactivity per cell and then frozen for several days to accumulate radiation damage. The overall viability of the population was reduced by 5- to 50-fold. Four consecutive selection cycles were carried out. The surviving cells were screened by replica plating-fluorography for clones showing decreased incorporation of fucose into trichloroacetic acid-insoluble macromolecules. Considerable enrichment for cells deficient in fucose uptake or incorporation into proteins (or both) was found in populations surviving the later selection cycles. Two mutant clones isolated after the fourth selection cycle had the same doubling time as the wild type, but contained only 30 to 40% as much fucose bound to proteins as the wild type. Sialic acid contents of the mutants and the wild type were similar. The mutants differed quantitatively and qualitatively from the wild type and from each other with respect to total glycoprotein profiles as visualized by sodium dodecyl sulfate gel electrophoresis. Differences were also found in resistances to cytotoxicity of lectins such as concanavalin A and wheat germ agglutinin.

scholarly journals Rapid cell surface appearance of endocytic membrane proteins in Chinese hamster ovary cells.

1981 ◽  
Vol 1 (3) ◽  
pp. 261-268 ◽  
Author(s):  
B Storrie ◽  
T D Dreesen ◽  
K M Maurey
Keyword(s):  
Membrane Proteins ◽  
Cell Surface ◽  
Chinese Hamster Ovary ◽  
Cho Cells ◽  
Gel Electrophoresis ◽  
Chinese Hamster Ovary Cells ◽  
Chinese Hamster ◽  
Surface Appearance ◽  
Ovary Cells ◽  
One Dimensional

Lactoperoxidase was used to selectively radiolabel endocytic membrane. CHO cells were incubated with enzyme at 37 degrees C for 10 min to permit lactoperoxidase internalization. Radioiodination was done at 4 degrees C. About 90% of the radioiodinated products pelleted at 100,000 X g. From 12 to 15 different electrophoretic species were detected by one-dimensional gel electrophoresis. When cells labeled by internalized lactoperoxidase were warmed to 37 degrees C, the incorporated radioactivity was lost in a biphasic manner with an overall t1/2 of approximately 20 h. Upon warming cells to 37 degrees C, the labeled species became sensitive to pronase or trypsin digestion. The increase in protease sensitivity was progressive over a 10- to 20-min period. Maximally 45% of the initially intracellular radiolabel could be released. A digest of exterior-radioiodinated cells released 50% of the incorporated radioiodine. These observations strongly suggest a rapid shuttling of approximately 90% of the radioiodinated membrane species initially present within the cell to the cell surface.

scholarly journals Isolation of a novel cell-attachment and spreading-promoting protein from human serum

1985 ◽  
Vol 227 (2) ◽  
pp. 421-427 ◽  
Author(s):  
M Vuento ◽  
M Korkolainen ◽  
P Kuusela ◽  
E Hölttä
Keyword(s):  
Affinity Chromatography ◽  
Human Serum ◽  
Isoelectric Focusing ◽  
Chinese Hamster Ovary ◽  
Gel Electrophoresis ◽  
Cell Attachment ◽  
Chinese Hamster Ovary Cells ◽  
Polyacrylamide Gel Electrophoresis ◽  
Chinese Hamster ◽  
Ovary Cells

A protein with potent cell-attachment and spreading-promoting activity was isolated from fibronectin-free human serum. The purification steps included affinity chromatography on heparin-agarose and preparative isoelectric focusing. The purified protein was homogeneous as judged from dodecyl sulphate/polyacrylamide-gel electrophoresis. It had an isoelectric point of 5.0 and an Mr of 52 000. The protein promoted the spreading of Chinese-hamster ovary cells to plastic in a manner similar to that observed with fibronectin.

Rapid cell surface appearance of endocytic membrane proteins in Chinese hamster ovary cells

1981 ◽  
Vol 1 (3) ◽  
pp. 261-268
Author(s):  
B Storrie ◽  
T D Dreesen ◽  
K M Maurey
Keyword(s):  
Membrane Proteins ◽  
Cell Surface ◽  
Chinese Hamster Ovary ◽  
Cho Cells ◽  
Gel Electrophoresis ◽  
Chinese Hamster Ovary Cells ◽  
Chinese Hamster ◽  
Surface Appearance ◽  
Ovary Cells ◽  
One Dimensional

Lactoperoxidase was used to selectively radiolabel endocytic membrane. CHO cells were incubated with enzyme at 37 degrees C for 10 min to permit lactoperoxidase internalization. Radioiodination was done at 4 degrees C. About 90% of the radioiodinated products pelleted at 100,000 X g. From 12 to 15 different electrophoretic species were detected by one-dimensional gel electrophoresis. When cells labeled by internalized lactoperoxidase were warmed to 37 degrees C, the incorporated radioactivity was lost in a biphasic manner with an overall t1/2 of approximately 20 h. Upon warming cells to 37 degrees C, the labeled species became sensitive to pronase or trypsin digestion. The increase in protease sensitivity was progressive over a 10- to 20-min period. Maximally 45% of the initially intracellular radiolabel could be released. A digest of exterior-radioiodinated cells released 50% of the incorporated radioiodine. These observations strongly suggest a rapid shuttling of approximately 90% of the radioiodinated membrane species initially present within the cell to the cell surface.

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