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Toxicity and Side Effects of Cell-Penetrating Peptides 251
11.2.1.2 Viability Assays
Cell viability assays are based on counting living active cells instead of damaged
or dead cells, which are counted in methods based on exclusion and leakage. These
viability assays estimate the functioning of whole cells, usually assuming the intactness
of the plasma membranes. Mainly, the activity of enzymes, pumps, and transporters
is used for measuring cellular function, even under unfavorable conditions.
11.2.1.2.1 Enzymatic Assays
Many enzymatic viability assays are based on the activity of cytosolic esterases that
convert neutral nonpolar esters of dyes that diffuse into the cells to colored or
fluorescent substances. The products, which are polar or poorly soluble, cannot leave
a cell via intact plasma membrane. The diacetyl derivative of fluorescein 27 was the
first and, for a long time, the most commonly used compound for measuring viability
enzymatically (Figure 11.1.d). An alternative compound for wider application is now
provided by acetoxymethyl ester of calcein. This substance has the advantages of
longer cellular retention time and lower pH sensitivity as compared to fluoresceinyl
derivatives.
The functionality of mitochondria is assessed in the MTT assay described in the
early eighties by Mosmann as a rapid colorimetric method for measuring cellular
growth and survival. 28 It is based on the cleavage of a yellow, water-soluble tetrazolium
dye, MTT, by succinate dehydrogenase (Figure 11.1.e). The tetrazolium ring
is cleaved only in active mitochondria, resulting in a purple, water-insoluble MTT
formazan product whose absorbency can be measured after dissolving the formed
crystals in an organic solvent. MTT cleavage occurs only in active cells; the reaction
is quick and no washing steps are required, which makes the method convenient
and popular for measuring cytotoxicity, proliferation, and activation. However, a
number of factors can significantly influence the cleavage of MTT, 29 such as cellular
uptake and metabolism of glucose, cellular concentration of the reduced pyridine
nucleotides NADH and NADPH, pH of media, and type of cell line. These parameters
must be considered when establishing MTT assay conditions.
Both methods — the retention of fluorescein and the MTT assay — have been
used to study effects of CPPs on cell viability 8,21,30 (see Table 11.2).
11.2.1.2.2 Uptake Assays
Maintenance of organelles and their specific environment is essential for cell survival.
Some dyes sensitive to minor pH changes can diffuse into cells and will
concentrate in acidic organelles, thereby reporting maintenance of organelle intactness
and functionality. Acridine orange and neutral red can be used to reveal and
assess the functioning of lysosomes. 31
The dye rhodamine 123 (R123), which is sensitive to changes in membrane
potential, concentrates in active mitochondria resulting in an intense fluorescence
signal (Figure 11.1.f). This enables detection of mitochondria membrane intactness
by the membrane potential and also visualization of these organelles. 32 Loss of R123
fluorescence is a rapid indicator of decreased cell viability and precedes the onset
of propidium iodide staining of the nuclei. New derivatives of Mitotracker ® and