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A method for incorporating macromolecules into adherent cells.

P L McNeil, R F Murphy, F Lanni

    The Journal of Cell Biology
    |April 1, 1984
    PubMed
    Summary
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    This study introduces scrape-loading, a straightforward technique for introducing macromolecules into mammalian cells. This method efficiently loads various molecules into diverse cell types, showing promise for cellular research.

    Area of Science:

    • Cell Biology
    • Biotechnology

    Background:

    • Efficiently introducing exogenous macromolecules into mammalian cells is crucial for various biological studies.
    • Existing methods may have limitations in terms of efficiency, cell type applicability, or molecule size.

    Purpose of the Study:

    • To develop and describe a simple, effective method for loading macromolecules into the cytoplasm of adherent mammalian cells.
    • To evaluate the efficiency, cell viability, and applicability of the new method across different cell types and macromolecules.

    Main Methods:

    • Developed a technique termed "scrape-loading" involving harvesting adherent cells, exposing them to labeled macromolecules, and then replating.
    • Assessed cell viability post-procedure and quantified macromolecule uptake using fluorescence microscopy.
    • Tested the method on fibroblasts, HeLa cells, macrophage-like cell line (1774A.1), and human neutrophils.

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    Main Results:

    • Scrape-loading achieved 50-60% cell viability immediately after the procedure, with 90% viability after 24 hours.
    • Approximately 40% of fibroblasts successfully incorporated fluorescently labeled dextrans, ovalbumin, or immunoglobulin-G.
    • Loading efficiency correlated with macromolecule concentration and molecular weight; an average of 10^7 dextran molecules (70,000 MW) were loaded per fibroblast.

    Conclusions:

    • Scrape-loading is a simple and effective method for introducing exogenous macromolecules into the cytoplasm of various adherent mammalian cell types.
    • The technique demonstrates broad applicability for loading diverse macromolecules, including proteins like actin analogs, into cells.
    • This method offers a valuable tool for cellular research requiring cytoplasmic delivery of macromolecules.