A method for incorporating macromolecules into adherent cells.
We describe a simple method for loading exogenous macromolecules into the cytoplasm of mammalian cells adherent to tissue culture dishes. Culture medium was replaced with a thin layer of fluorescently labeled macromolecules, the cells were harvested from the substrate by scraping with a rubber policeman, transferred immediately to ice cold media, washed, and then replated for culture. We refer to the method as "scrape-loading." Viability of cells was 50-60% immediately after scrape-loading and was 90% for those cells remaining after 24 h of culture. About 40% of adherent, well-spread fibroblasts contained fluorescent molecules 18 h after scrape-loading of labeled dextrans, ovalbumin, or immunoglobulin-G. On average, 10(7) dextran molecules (70,000-mol wt) were incorporated into each fibroblast by scrape-loading in 10 mg/ml dextran. The extent of loading depended on the concentration and molecular weight of the dextrans used. A fluorescent analog of actin could also be loaded into fibroblasts where it labeled stress fibers. HeLa cells, a macrophage-like cell line, 1774A.1, and human neutrophils were all successfully loaded with dextran by scraping. The method of scrape-loading should be applicable to a broad range of adherent cell types, and useful for loading of diverse kinds of macromolecules.
D000818:Animals; D000941:Antigens; D001692:Biological Transport; D002448:Cell Adhesion; D002470:Cell Survival; D003911:Dextrans / Q000378:metabolism*; D016650:Fluorescein-5-isothiocyanate / Q000031:analogs & derivatives; D005452:Fluoresceins / Q000378:metabolism*; D005456:Fluorescent Dyes; D006367:HeLa Cells / Q000502:physiology; D006801:Humans; D007074:Immunoglobulin G / Q000378:metabolism*; D007700:Kinetics; D008264:Macrophages / Q000502:physiology*; D051379:Mice; D008970:Molecular Weight; D009504:Neutrophils / Q000502:physiology*; D010047:Ovalbumin / Q000378:metabolism*; D013861:Thiocyanates
D000941:Antigens; D003911:Dextrans; D005452:Fluoresceins; D005456:Fluorescent Dyes; D007074:Immunoglobulin G; D013861:Thiocyanates; C015219:fluorescein isothiocyanate dextran; D010047:Ovalbumin; D016650:Fluorescein-5-isothiocyanate