Techniques for introducing normally impermeant macromolecules into the living cell-referred to as "cell-loading techniques"-are useful in a variety of settings for the cell biologist. Microinjection is probably the most commonly used technique for introducing fluorescent probes, fluorescently tagged proteins, and antibodies into living cells for short-term studies of cell physiology and protein location and function. It is, however, not the only technique available, nor the easiest or least expensive to implement. Among the alternatives are several closely related techniques that, like microinjection, rely on the cell's ability to reseal a mechanically induced plasma membrane disruption created in order to gain temporary access to cell cytosol. Four such techniques are described in this unit: scrape loading, scratch loading, bead loading, and syringe loading. Unlike microinjection, these techniques allow one to rapidly load (in a matter of minutes) thousands or even many millions of many types of mammalian cells with normally impermeant molecules, and so to facilitate quantitative analyses of the effect of loading.
|Original language||English (US)|
|Journal||Current protocols in cell biology / editorial board, Juan S. Bonifacino ... [et al.]|
|Publication status||Published - Jan 1 2001|
ASJC Scopus subject areas
- Cell Biology