Fusion of Mouse Myeloma and Spleen Cells

Yelton, Dale E.; Diamond, Betty; Kwan, Sau‐Ping; Scharff, Matthew D.

doi:10.1007/978-3-642-67448-8_1

Cited by 45 publications

(24 citation statements)

References 17 publications

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“…In the absence of affinity targeting, PEG-mediated fusion of lymphocytes occurs at low frequencies, variously reported at 10-4 to 10-5 (1,(21)(22)(23). Similarly, our experiments show that random fusions of untargeted vesicles to lymphocytes are rarely observed with PEG exposures at concentrations and for durations consistent with preserving cell viability (Tables 1-3).…”

Section: Discussionsupporting

confidence: 82%

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: Discussionsupporting

confidence: 82%

Immunospecific vesicle targeting facilitates microinjection into lymphocytes.

Godfrey¹,

Doe²,

Wofsy³

1983

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

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“…Formaldehyde-fixed, heat-killed Stuphylococcus aureus (Cowan I) was obtained from Calbiochem and was used as an immunoadsorbant. The &azaguanine-resistant murine myeloma cell line P3-X63-Ag.Ul (P3Ul) (Yelton et al, 1978) was obtained from Osaka University Medical School. Catalytic subunit of CAMP-dependent protein kinase was partially purified from rat heart by the method of Reimann and Beham (1983).…”

Section: Methodsmentioning

confidence: 99%

Monoclonal antibodies against rat brain protein kinase C and their application to immunocytochemistry in nervous tissues

Kitano¹,

Hashimoto²,

Kikkawa³

et al. 1987

J. Neurosci.

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Three monoclonal antibodies were prepared against rat brain soluble protein kinase C. Two of the antibodies, CKI-97 (IgG2b subclass) and CKII-90 (IgG1 subclass), showed weak binding to native protein kinase C. The third antibody, CKI-33 (IgG2b subclass), showed no binding. However, the mixture of CKI-97, CKII-90, and CKI-33 exhibited much stronger binding activity to this protein kinase than any of the antibodies alone. Although none of these antibodies showed protein kinase C-neutralizing activity, Western blot analysis indicated that these antibodies reacted specifically with protein kinase C, presumably its subspecies, that is present predominantly in nervous tissues. Immunocytochemical studies shows that these antibodies can be used for identification of this enzyme in nervous tissues. In rat Purkinje cells, the immunoreactive material was present throughout the cytoplasm, including dendrites and axons, but was poorly represented in the cell nucleus. In cerebellum, the localization of protein kinase C appears to be very similar to that of cGMP-dependent protein kinase.

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“…NCC-LU-81 was established after immunization of BALB/c mice with cultured cells derived from Lu65 tumors. The fusion was performed with P3-X63-AG8-U1 cells (12) using polyethylene glycol 4000. The hybridomas were grown and selected as described (13) by immunohistological screening of small tissue sections of three types of lung cancers (adenocarcinoma, squamous cell carcinoma, and giant cell carcinoma) and normal lung tissues.…”

Section: Methodsmentioning

confidence: 99%

Blood group A cross-reacting epitope defined by monoclonal antibodies NCC-LU-35 and -81 expressed in cancer of blood group O or B individuals: its identification as Tn antigen.

Hirohashi¹,

Clausen²,

Yamada³

et al. 1985

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

162

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Two monoclonal antibodies, NCC-LU-35 and NCC-LU-81, have been established after immunization of mice with membrane preparations of human lung cancer Lu65 tumor xenograft cells grown in vivo and intact cells cultured in vitro, respectively. These two antibodies react specifically with a majority of human adenocarcinomas, irrespective of the host's blood group ABO status, as well as with normal tissues and erythrocytes of blood group A individuals. The antigenicity is associated with a high molecular weight mucin-like glycoprotein separated by gel filtration of Lu65 tumor extracts. The epitope of the mucin-like glycoprotein has been identified as a-N-acetylgalactosaminyl residue directly linked O-glycosidically to serine or threonine residues of polypeptides. This epitope was serologically detected several years ago and given the name Tn. Our identification of the epitope is based on the following results: (i) The antigen is sensitive to a-Nacetylgalactosaminidase, but not to sialidase or a-fucosidase.(ii) Various mono-and difucosyl A determinants, either type 1 or type 2 chain, cross-react with both antibodies. (ii) The reactivity with both antibodies can be created by treatment of glycophorin A of normal erythrocytes with sialidase followed by 13-galactosidase. Monoclonal antibodies NCC-LU-35 and NCC-LU-81 were established after immunization of mice with lung carcinoma Lu65 cells. The antigen defined by both antibodies is not expressed in various normal tissues of blood group B and 0 hosts, but it is strongly expressed in various adenocarcinomas derived from blood group B and 0 as well as A hosts.

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Fusion of Mouse Myeloma and Spleen Cells

Cited by 45 publications

References 17 publications

Immunospecific vesicle targeting facilitates microinjection into lymphocytes.

Immunospecific vesicle targeting facilitates microinjection into lymphocytes.

Monoclonal antibodies against rat brain protein kinase C and their application to immunocytochemistry in nervous tissues

Blood group A cross-reacting epitope defined by monoclonal antibodies NCC-LU-35 and -81 expressed in cancer of blood group O or B individuals: its identification as Tn antigen.

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