dead cells are stained whereas viable cells exclude the
stain. Alternatively, use an automated cell counter.
3.2. Plasma cell permeabilization analyzed by fluorometry, flow
cytometry or high content microscopy
Loss of plasma membrane integrity can be assessed by using
cell-impermeant dyes. Once the integrity of the plasma membrane
is lost, the dye can enter the dead cell. A variety of DNA-binding,
impermeant fluorescent dyes exist, such as propidium iodide (PI,
MW 668.4 Da), 7-amino actinomycin D (7-AAD) or the Sytox
probes (Life Technologies, MW 600 Da). The fluorescence intensity
of these dyes increases markedly after DNA binding, thus
eliminating the need for time-consuming washing steps. This fluorescence
intensity shift upon DNA binding can be exploited in
assays done in microtiter plates, as only dead cells are intensely
fluorescent. A major advantage of biophysical assays over enzymatic
assays is that they are not prone to expression and activity
regulation as in the case of, for example, MTT assays (see Section
2.2). A disadvantage of the fluorescent-probe-based biophysical
assays is the possible interference of fluorescent (experimental)
compounds with the measurement.