A method for incorporating macromolecules into adherent cells.

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 .Methods for loading macromolecules into cytoplasm are essential to studies in several recent and evolving fields of eucaryotic cell and molecular biology. Transformation ofcells with exogenous DNA, in vivo studies of the location and activity of cytoskeletal proteins and other macromolecules in living cells by fluorescent analog cytochemistry, and the analysis of cytoplasmic degradation of proteins all depend on the incorporation of large molecules into living cells .Certain characteristics are desirable of any method for loading macromolecules into cells. The method should be simple, applicable to diverse cell types, and capable of loading a wide range of macromolecule species. A significant proportion of cells in a population should be loaded with the macromolecule and to a measurable extent. The macromolecules should be introduced, initially, into the cell's cytoplasm only. Cell function, morphology, and viability should not be extensively compromised by the loading method.Several methods are now available for loading exogenous macromolecules into cell cytoplasm . Microneedles are used to inject diverse substances into individual cells (8; see 6 for a review) . Briefexposure to a hypotonic medium loads protein in that medium into the cytoplasm of a cell population (2) . 1556Hyposomotic shock ofpinosomes containing dextran or protein releases most of these substances into the cytoplasm of cells (14). Liposomes and red blood cells containing trapped substances can, after fusion with host cells, deliver such trapped substances into host cytoplasm (17,7,15). In addition, high voltage electric impulses enhance uptake of DNA into cells (13).No single one ofthese methods now available fulfills all the criteria li...

Activation of mitogen-activated protein kinase is necessary but not sufficient for proliferation of human thyroid epithelial cells induced by mutant Ras

“…Since retroviral vectors only target cells already in the cell cycle, however, it was formally possible that responsiveness to Ras activation was limited to the tiny sub-population of thyroid cells which are normally cycling in standard in vitro conditions (Wynford-Thomas, 1993). Before proceeding to an analysis of mechanism, therefore, we ®rst addressed this issue of population homogeneity, by using two approaches which target cells independent of their proliferative state, namely scrape-loading (transient permeabilization) (McNeil et al, 1984;Morris et al, 1989) and micro-injection.…”

Innate recognition of bacteria by a macrophage cytosolic surveillance pathway

“…Using an LLO-independent method, scrape-loading of bacterial products into HeLa cells, yielded similar results. Scrape-loading relies on temporary disruption of host cell membranes on scraping of an established monolayer to allow access of macromolecules into the host cytosol (20). Scrape-loading LTA or LPS at concentrations that activate signaling through TLRs in macrophages did not result in cytokine induction in HeLa cells, whereas scrape-loaded E. coli or B. subtilis extracts strongly induced cytokine gene expression (data not shown).…”

Dissecting the Role of Rho-mediated Signaling in Contractile Ring Formation

“…HeLa cells were loaded with C3 exoenzyme (Calbiochem) by the scrapeloading method (McNeil et al, 1984) with slight modification. Briefly, HeLa cells in a 35-mm dish (1 ϫ 10 5 cells) were scraped in 100 l of 0.1 mg/ml C3 dissolved in phosphate-buffered saline (PBS) with a rubber policeman and incubated at room temperature for 10 min.…”

“…It is reported that C3-microinjected HeLa cells frequently detach from the dish or fail to initiate anaphase (O'Connell et al, 1999). To avoid these problems, we used the scrape-loading method to deliver C3 to HeLa cells (McNeil et al, 1984). The advantage of the scrapeloading method enabled us to analyze a large number of C3-loaded cells without decreasing cell viability.…”