Improved fusion methods. IV. Technical aspects

Westerwoudt, Regine J.

doi:10.1016/0022-1759(85)90031-6

Cited by 51 publications

(26 citation statements)

References 108 publications

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“…For cell-to-cell fusion we have chosen the polyethylene glycol (PEG) method [16,26,35]. Another frequently used fusion start Seed 2'106 cells in a plastic petrl dish ( ~'11 cm); Rrow in MEM medium with 10 0~ FCS (pH 7.4) for 3 days to subconfluency; cell-free area in the subconfluent monofayer is appr.…”

Section: Methods Of Cell Fusionmentioning

confidence: 99%

Fusion of cultured dog kidney (MDCK) cells: I. Technique, fate of plasma membranes and of cell nuclei

et al. 1989

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The evaluation of the intracellular signal train and its regulatory function in controlling transepithelial transport with electrophysiological methods often requires intracellular measurements with microelectrodes. However, multiple impalements in epithelial cells are hampered by the small size of the cells. In an attempt to avoid these problems we fused cells of an established cell line. Madin Darby canine kidney cells, originally derived from dog kidney, to "giant" cells by applying a modified polyethylene glycol method. During trypsin-induced detachment from the ground of the petri dish, individual cells grown in a monolayer incorporate volume and mainly lose basolateral plasma membrane by extrusion. By isovolumetric cell-to-cell fusion, spherical "giant" cells are formed within 2 hr. During this process a major part of the individual cell plasma membranes is internalized. Over three weeks following cell plasma membrane fusion degradation of single cell nuclei and cell nuclear fusion occurs. We conclude that this experimental approach opens the possibility to investigate ion transport of epithelia in culture by somatic cell genetic techniques.

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Section: Methods Of Cell Fusionmentioning

confidence: 99%

Fusion of cultured dog kidney (MDCK) cells: I. Technique, fate of plasma membranes and of cell nuclei

et al. 1989

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“…Next, they were fused using 50% (w/v) polyethylene glycol 1500 (Boehringer, Mannheim, Germany) according to standard procedures [34,38]. After fusion, the cells were seeded in two 96-well plates in a double selection medium; DMEM+FBS supplemented with G418 (2 mg/ml) and HAT (100 pH hypoxanthine, 0.4 pñ aminopterin and 16 pK thymidine).…”

Section: Cell Viabilitymentioning

confidence: 99%

Enhanced Transfection of a Bacterial Plasmid into Hybridoma Cells by Electroporation: Application for the Selection of Hybrid Hybridoma (Quadroma) Cell Lines

Bos¹,

Nieuwenhuizen²

1992

Hybridoma

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A procedure was investigated to transduce a bacterial plasmid containing a specific drug resistance marker (pSV2-neo), into a hybridoma cell line using electroporation. The effect of several buffers and the form of plasmid DNA (circular or linearized) on the stable transfection frequency were examined. When complete cell culture medium (DMEM) was used as electroporation buffer, we observed a two-fold increase in post-pulse viability and a ten- to thirty-fold increase in the transfection frequency of pSV2-neo, as compared with HEPES buffered 0.15 M sodium chloride. Supplementing DMEM with fetal bovine serum (DMEM + FBS) had some beneficial effect on post-pulse viability of the cells after electroporation, but did not markedly increase stable transfection frequency as compared with DMEM alone. Furthermore, with DMEM + FBS, the intact plasmid was transfected as effectively as linearized PSV2-neo. However, when using HEPES buffered saline, the transfection frequency of pSV2-neo increased two-fold after linearization as compared with intact plasmid. The drug resistance was used successfully as a marker for the selection of hybrid hybridoma (quadroma) cell lines after fusing two different hybridoma cell lines, producing anti-fibrin and anti-plasminogen activator antibodies respectively. The quadroma cells produced bispecific antibodies that are capable of accumulating plasminogen activator on a fibrin surface.

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“…In this brief review we shall attempt to highlight some of the most recent developments in hybridoma technology which seem to offer practical advantages -or which at least show a potential for practical use in the near future. Readers who seek more comprehensive information on current hybridoma technology and monoclonal antibody applications are encouraged to examine any of a number of excellent reviews (Milstein, 1982;Reading, 1982;Denis et al, 1983;Schonherr and Houwink, 1984;Sikora, 1984;KShler, 1985;Westerwoudt, 1985;DePinho et al, 1986). Those who seek more information on how to generate hybridomas should consult articles detailing that technology (Oi and Herzenberg, 1980;Galfr6 and Milstein, 1981;KShler, 1981;Bastin et al, 1982;Lane et al, 1982;Nakamura et al, 1982;Campbell, 1984;Goding, 1986).…”

Section: Introductionmentioning

confidence: 99%

Hybridoma technology: new developments of practical interest

Samoilovich

Dugan

Macario

1987

Journal of Immunological Methods

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ScopeThis brief review has a limited scope. It focuses on technical developments of practical interest concerning construction of hybridomas and their expansion for large scale production of monoclonal antibodies.Some of the procedures surveyed are not yet routinely applied but are promising. Highlighted are tactics to improve fusion efficiency and production of antibodies in vitro, the use of chemically defined culture media, and attempts at generating antibodies other than murine, especially human.Beginners in hybridoma technology and those planning to use it soon should be aware of current trends beyond the classical technology. These trends should be carefully considered before making basic decisions, for example what cell line to use as a fusion partner.It should be clear that new avenues are being explored with success. Mouse and rat hybridomas are no longer the only alternatives. Monoclonal antibodies of other species, e.g., human, are within the realm of the possible. The same can be said for

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Improved fusion methods. IV. Technical aspects

Cited by 51 publications

References 108 publications

Fusion of cultured dog kidney (MDCK) cells: I. Technique, fate of plasma membranes and of cell nuclei

Fusion of cultured dog kidney (MDCK) cells: I. Technique, fate of plasma membranes and of cell nuclei

Enhanced Transfection of a Bacterial Plasmid into Hybridoma Cells by Electroporation: Application for the Selection of Hybrid Hybridoma (Quadroma) Cell Lines

Hybridoma technology: new developments of practical interest

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