A method for incorporating macromolecules into adherent cells

P. L. McNeil, R. F. Murphy, F. Lanni, D. L. Taylor

Research output: Contribution to journalArticle

216 Scopus citations

Abstract

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, 107 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, J774A.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.

Original languageEnglish (US)
Pages (from-to)1556-1564
Number of pages9
JournalJournal of Cell Biology
Volume98
Issue number4
DOIs
StatePublished - 1984

ASJC Scopus subject areas

  • Cell Biology

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    McNeil, P. L., Murphy, R. F., Lanni, F., & Taylor, D. L. (1984). A method for incorporating macromolecules into adherent cells. Journal of Cell Biology, 98(4), 1556-1564. https://doi.org/10.1083/jcb.98.4.1556