Red cell-mediated microinjection of macromolecules into monolayer cultures of mammalian cells

Mercer, W E; Terefinko, Dominik; Schlegel, Robert

doi:10.1016/0309-1651(79)90039-0

Cited by 20 publications

(5 citation statements)

References 10 publications

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“…Values of less than 2% hemoglobin release during digestion were consistently obtained. In addition, ghosts loaded with fluorescein-conjugated bovine serum albumin (Mercer et al, 1979) did not leak this protein, even upon phospholipase digestion. General membrane integrity was also routinely assessed by microscopic examination.…”

Section: Methodsmentioning

confidence: 95%

Phospholipid asymmetry in human erythrocyte ghosts

Williamson

Algarin

Bateman

et al. 1985

Journal Cellular Physiology

122

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Using phospholipase digestion and the fluorescent probe merocyanine 540 the maintenance of phospholipid asymmetry in the plasma membrane of human erythrocyte ghosts was investigated. Digestion with phospholipase A2 indicated that ghosts prepared in the presence of Mg++ as the only divalent cation retained the normal phospholipid asymmetry characteristic of intact erythrocytes. These ghosts, like normal erythrocytes, also failed to stain with merocyanine 540. However, the presence of as little as 5-10 microM Ca++ during ghost preparation resulted in ghosts in which lipid asymmetry had been abolished, as indicated by phospholipase digestion. Moreover, these ghosts stained with merocyanine 540. In contrast to ghosts, intact erythrocytes treated with ionophore required millimolar levels of Ca++ ions to disrupt membrane lipid asymmetry. To discover the reason for this difference in behavior between ghosts and intact cells, ghosts were prepared from preswollen cells using only small volumes of buffer for lysis. These experiments demonstrated that as the cellular contents of erythrocytes are diluted, the asymmetric arrangement of phospholipids becomes more sensitive to disruption by Ca++.

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Section: Methodsmentioning

confidence: 95%

Phospholipid asymmetry in human erythrocyte ghosts

Williamson

Algarin

Bateman

et al. 1985

Journal Cellular Physiology

122

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“…Ghosts prepared by the dilution method were loaded with fluoresceinated BSA (fl-BSA) by including it in the hypotonic lysing buffer (Mercer et al, 1979). Ghosts or intact erythrocytes were labelled with 51Cr by incubating as a 20% suspension in Hanks' buffer containing 60 pgiml of Na2Cr04 (250-475 pCiIpg at the time of use) for 30 minutes at 37°C.…”

Section: Methodsmentioning

confidence: 99%

“…Symmetric or asymmetric ghosts were prepared from human erythrocytes and their distribution of phospholipids verified by fluorescence staining as described in the preceding paper (Williamson et al, 1985). Ghosts prepared by the dilution method were loaded with fluoresceinated BSA (fl-BSA) by including it in the hypotonic lysing buffer (Mercer et al, 1979). Ghosts or intact erythrocytes were labelled with 51Cr by incubating as a 20% suspension in Hanks' buffer containing 60 pgiml of Na2Cr04 (250-475 pCiIpg at the time of use) for 30 minutes at 37°C.…”

Section: Methodsmentioning

confidence: 99%

Membrane phospholipid asymmetry as a factor in erythrocyte‐endothelial cell interactions

Schlegel¹,

Prendergast²,

Williamson³

1985

Journal Cellular Physiology

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The tendency of human erythrocytes to adhere to vascular endothelial cells was assessed as a function of the transbilayer distribution of the phospholipids of the erythrocyte membrane, using erythrocyte ghosts in which transbilayer lipid arrangement was manipulated by varying the conditions under which the ghosts were prepared. By two different assays, ghosts with symmetric lipid bilayers adhered strongly to monolayers of cultured endothelial cells, whereas ghosts with normal asymmetric membranes, like normal erythrocytes, did not. These results provide direct evidence that changes in phospholipid asymmetry can alter the tendency of erythrocytes to adhere to endothelial cells, and therefore imply that transbilayer phospholipid arrangement may influence the behavior of erythrocytes in the circulatory system and may contribute to the formation of microvascular occlusions.

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“…Fibroblast microinjection was performed essentially as described by Mercer et al (18). One-half milliliter of fusion medium, autoclaved with Pi/NaCl, was overlayed on an aspirated well.…”

Section: Methodsmentioning

confidence: 99%

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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Red cell-mediated microinjection of macromolecules into monolayer cultures of mammalian cells

Abstract: Phytohemagglutinin has been used to enhance the red cell-mediated microinjection of macromolecules into monolayer cultures of mammalian cells. The simple procedure described should promote the wider use of this microinjection technique.

Cited by 20 publications

References 10 publications

Phospholipid asymmetry in human erythrocyte ghosts

Phospholipid asymmetry in human erythrocyte ghosts

Membrane phospholipid asymmetry as a factor in erythrocyte‐endothelial cell interactions

Immunospecific vesicle targeting facilitates microinjection into lymphocytes.

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