Affinity targeting of membrane vesicles to cell surfaces

Godfrey, William L.; Doe, Barbara; Wallace, Ellen F.; Bredt, Barry M.; Wofsy, Leon

doi:10.1016/0014-4827(81)90306-2

Cited by 35 publications

(14 citation statements)

References 12 publications

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“…Fab anti-MIg was modified with tetramethylrhodamine isothiocyanate (Baltimore Biological Laboratory) as described (11). Bovine serum albumin (Pentex, Kankakee, IL) and avidin were made fluorescent with fluorescein isothiocyanate (Baltimore Biological Laboratory) as described (9). Bovine serum albumin (10 mg) was modified with 1 mCi (1 Ci = 3.7 X 10'°Bq) of Naa25I (New England Nuclear) by the IODO-BEAD method (Pierce).…”

Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

“…Biotin derivatives of antibody were prepared by reaction of the proteins with biotin-N-hydroxysuccinimide (14) as described by Heggeness and Ash (15). The pyridyldithiopropionate derivative of avidin was prepared by allowing avidin (Sigma) to react with N-hydroxysuccinimidyl 3-(2-pyridyldithio)propionate (SPDP) (Pharmacia) as described (9). Fab anti-MIg was modified with tetramethylrhodamine isothiocyanate (Baltimore Biological Laboratory) as described (11).…”

Section: Methodsmentioning

confidence: 99%

“…PEG-mediated microinjection can be enhanced by agglutinating vesicles to cells with phytohemagglutinin (6,8). Our laboratory has developed protocols for the efficient, antibody-directed targeting of vesicles to cell surface antigens (9). In this paper, we describe the use of vesicle targeting with PEG-mediated fusion to promote the microinjection of selected cell types, particularly of lymphocytes and lymphoid cell lines.…”

mentioning

confidence: 99%

“…Avidin or Fab' anti-MFab was coupled to human RBC by method III or method II, respectively, of Godfrey et al (9). RBC, or protein-coupled RBC, were made into fluorescent sealed ghosts by the method of Bodemann and Passow (16) as modified by Godfrey et al (9). Ghosts, avidin-coupled ghosts (avidin-ghosts), and Fab' anti-MFab-coupled ghosts (Fab'-ghosts) were resuspended at 10% (vol/vol) in phosphate-buffered saline (Pi/NaCl; 0.002 M NaH2PO4/0.008 M Na2HPO4/0.15 M NaCl, pH 7.5) or Dulbecco's modified Eagle medium (DME medium; GIBCO).…”

mentioning

confidence: 99%

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Immunospecific vesicle targeting facilitates microinjection into lymphocytes.

Godfrey¹,

Doe²,

Wofsy³

1983

Proc. Natl. Acad. Sci. U.S.A.

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Antibody-directed targeting of vesicles to cells dramatically enhances polyethylene glycol-mediated fusion and microinjection. Sealed erythrocyte ghosts, containing fluorescent bovine serum albumin, were targeted to murine spleen and thymus cells, and to lymphocyte, monocyte, and fibroblast cell lines. In all cases, targeted cell populations showed substantial levels of microinjection, whereas populations treated with the fusogen in the absence of targeting were not significantly microinjected. To achieve attachment of vesicles to selected cells, the cells were first labeled with biotin-modified antibody then treated with sealed ghosts prepared from avidin-coupled erythrocytes. This procedure should prove useful when the injection of specific cell populations is desired, or with cell types such as lymphocytes that are difficult to fuse, or when the use of limited reagents necessitates high injection efficiencies.Microinjection allows the introduction into cells of biologically interesting molecules so that their properties and effects on cell function can be studied. Although microinjection protocols have been used to study a number of important questions in cell biology (1-4), their applications have been restricted by inherent limitations. Mechanical injection (5) can be used to introduce discrete volumes of reagent into cells, but it is limited by the number and type of cells that can be treated (1). Vesicle-mediated protocols, using fusogens such as polyethylene glycol (PEG) or Sendai virus, can be used to inject large numbers of cells (1), but fusion efficiencies are generally low and selectivity is lacking.We, as others (6), have reasoned that bringing two membranes into close apposition should increase the probability that they will fuse, although contact per se between two membranes does not generally lead to fusion (7). PEG-mediated microinjection can be enhanced by agglutinating vesicles to cells with phytohemagglutinin (6, 8). Our laboratory has developed protocols for the efficient, antibody-directed targeting of vesicles to cell surface antigens (9). In this paper, we describe the use of vesicle targeting with PEG-mediated fusion to promote the microinjection of selected cell types, particularly of lymphocytes and lymphoid cell lines. The frequency of vesicle-lymphocyte fusions in these experiments is about 104 times higher than values generally observed for PEG-mediated lymphocyte fusion. MATERIALS AND METHODSAntibodies. Goat anti-mouse immunoglobulin (anti-MIg) was raised as described (10). Anti-mouse cell surface (anti-MCS) was prepared by injecting rabbits with DBA/2 spleen and lymph node cells in complete Freund's adjuvant. Rabbit anti-mouse Fab fragment (anti-MFab) was generously donated by A. Good (University of California, Berkeley). All three antisera were passed over DEAE-Sephacel (Pharmacia). Fab fragment of antiMIg (11) and the affinity-purified F(ab')2 fragment of anti-MFab (9) were prepared as described. Two monoclonal antibodies were obtained from Becton Dickinson: mouse ant...

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Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%