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.
Article| April 01 1984
A method for incorporating macromolecules into adherent cells.
P L McNeil
R F Murphy
D L Taylor
Online ISSN: 1540-8140
Print ISSN: 0021-9525
J Cell Biol (1984) 98 (4): 1556–1564.
P L McNeil, R F Murphy, F Lanni, D L Taylor; A method for incorporating macromolecules into adherent cells.. J Cell Biol 1 April 1984; 98 (4): 1556–1564. doi: https://doi.org/10.1083/jcb.98.4.1556
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