Scrape loading

Selected entries from Methods in Enzymology [vol, page(s)] Technique and sample preparation, 207, 159 outside-out configuration, 207, 10 amplifiers, 238, 153, 310 scrape loading,... 

Scrape and scratch loading techniques Scrape loading. By scrape loading, adherent cells are scraped off their substratum with a cell scraper in the presence of the macro-molecule(s) (up to 500 kD mol weight) to be loaded (McNeil et al 1984). Thus loaded cells are obtained in suspension, and have to be replated and allowed to spread before microscopic observation. 

Scratch loading. Scratch loading is a variation of the scrape loading technique (Swanson and McNeil, 1987). An artificial wound is made on a cell monolayer with a sharp instrument in the presence of a solution of the molecule(s) to be loaded (up to 150 kD mol weight). Cells lining the edge of the wound are loaded. 

Figure 7 Utility of the scrape loading/dye transfer assay to detect a toxin/toxicant s ability to modulate gap junctional intercellular communication. The last two images (6ngmr TPA) illustrate both the epifluorescent and phase images, respectively, of the same cells to show that the complete inhibition of GJIC was not associated with the death of cells.

Figure 8 Use of the scrape loading/dye transfer assay to screen for agents that can prevent inhibition of gap junction function.

Fechheimer M, Boylan JF, Parker S, Sisken JE, Patel GL, Zimmer SG (1987) Transfection of mammalian cells with plasmid DNA by scrape loading and sonication loading. Proc Natl Acad Sci U S A 84 8463-8467... 

Sonnemann et al. (4) used both scrape loading and electroporation to successfully load cells with fura-2-dextran and measure [Ca ], changes in response to 0.1-1 pM cAMP. The results were in conflict to findings reported previously by Schlatterer et al. (5, 24), who scrape loaded cells with fura-2-dextran and reported no detectable change in [Ca +], after stimulation with submicromolar concentrations of cAMP. These contradictions may reflect the technical difficulties which are encountered when loading Dictyostelium cells with fluorescent Ca -sensitive indicators using either of these two methods. 

Scrape Loading of Cells with Fura-2 Dextran... 

Microinjection of Proteins into Somatic Cells Needle Microinjection and Scrape Loading... 

A rubber policeman for scrape loading was purchased from Fisher Scientific (Cat. No. 14-105A). 

Compared with needle microinjection, the amount of delivery varies more extensively among scrape-loaded cells. The yield of injected cells and the extent of cell damage also vary among cell types. Cell damage often manifests as the appearance of large vacuoles. 

The efficiency of scrape loading varies as a function of molecular weight (McNeil etai, 1984). 

Some molecules may not be compatible with the balanced salt solution required for scrape loading. 

Quantification of cell-to-cell communication. Flow cytometry meikes it easy to quantify cell-to-cell communication. As an example work of Kavanagh may be cited. Cells are scrape-loaded with Lucifer yellow with or without rhodamine labelled dextran. The transfer of the two dyes between donor and recipient cells can be studied easily using two colour fluorescence flow cytometry. 

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