Although it is possible to assess membrane integrity using fluorochrome-labeled antibodies that will react only with biomolecules confined to the cytoplasm or organelles, it is generally done using dyes that react with internal components (e.g., DNA) but that are normally excluded from cells with an intact plasma membrane. The DNA-binding dyes, 7-AAD (7-aminoactinomycin D) and PI, have long been used to identify cells with permeable membranes, but the newer DNA stains such as SYBR®-14, SYTOX GREEN®, YOYO®-1, and YO-PRO™-1 (Invitrogen) can also be used. Alternatively, cells can be stained with dyes that are membrane-permeant and that are retained inside live, but not dead, cells. These dyes are often used as their acetoxy-methyl (AM) esters, which diffuse rapidly into cells (at 37°C), where they can be hydrolyzed quickly by ubiquitous nonspecific esterases to yield intracellular fluorescent dye concentrations of more than 25 pmol/L within 10 to 60 min (22). Because the AM esters are poorly soluble in water, they are normally dissolved first in dimethyl-sulphoxide as a 1-10 mmol/L stock solution and then added to the medium at concentrations in the range of 1-25 pmol/L. Examples of dyes that are retained after hydrolysis, and that can be monitored in the fluorescence channel used for fluorescein, include fluorescein diacetate, 2',7'-bis(2-carboxyethyl)-5,6-carboxy-fluorescein AM ester (BCECF-AM) (Fig. 10A), CFDA, and calcein-AM, which is better retained than the others. Fura-Red is a similar dye that can be detected in the fluorescence channel used routinely for PE. Target cells can also be loaded with these dyes for use in lymphocyte cytotoxicity assays.
On occasion, it may be necessary both to assess the viability of cells in a sample and to fix them for subsequent analysis. This can be done if the cells are incubated with ethidium monoazide before fixation. Ethidium monoazide cannot enter cells with intact plasma membranes but will enter permeable cells and attach covalently to any nucleic acid if photoactivated. Consequently, after deliberate photoactivation in vitro, only the dead cells with damaged membranes (and not the viable cells) will be labeled and fluoresce. Alternatively, cells can be incubated with Tri-Color®-labeled streptavidin (streptavidin coupled to the tandem dye PE-Cy™5), which will specifically and irreversibly enter dead cells and cannot be removed by washing, permea-bilization, or fixation (23).
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