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Toxicity and Side Effects of Cell-Penetrating Peptides 249
f) R123,
Mitotracker
e) MTT
MT
d) Fluorescein DA
(FDA)
MTT formazan
N
(b)
FDA Fluorescein
g) Lysotracker
FIGURE 11.1b Methods used for estimating effects on cell viability. (d) Nonfluorescent,
nonpolar fluorescein diacetate (FDA) diffuses into cells across the plasma membrane. The
cellular esterases convert FDA into the fluorescent and polar fluorescein retained inside living
cells. (e) Soluble MTT is converted into water-insoluble formazan crystals by mitochondrial
enzymes of living cells. (f) R123 and Mitotracker ® concentrate into mitochondria and
(g) Lysotracker ® into lysosomes of viable cells only. (N = nucleus; MT = mitochondria; LYS =
lysosomes.)
intercalating dyes like ethidium bromide and, especially, propidium iodide
(Figure 11.1.b), which is better suited to fluorescence microscopy, flow cytometry,
and cell-sorting techniques, have gained more popularity. 18,19 New derivatives of
propidium iodide (PI) are less cell-permeable and practically nonfluorescent when
unbound to DNA, thus enabling a more sensitive detection system.
Dye exclusion assays are cheap, easy to perform, and well established over
decades. However, these methods tend to overestimate viability, since cells unable
to attach and proliferate (i.e., nonviable) may exclude the dye. Exclusion of trypan
blue 20 (see Table 11.1) and propidium iodide 21 have been used for evaluation of CPPinduced
toxicity.
11.2.1.1.2 Cytoplasmic Leakage
Another strategy for detection of cell plasma membrane damages is to estimate the
outflow of cytosolic enzymes or cell-introduced probes. 22 The release of lactate
dehydrogenase (LDH) into cell culture media 23 is one of the most traditional and
frequently used leakage assays. LDH activity is measured by the conversion of NAD
to NADH in the presence of excess lactate substrate. Release of LDH has been used
in different toxicity assays and enables assessment of accumulated leakage over a
remarkable time period. The major drawback with this method is the latency of
LDH, since leakage of a high molecular weight enzyme is relatively slow as compared
to dansyl lysine or PI nuclear staining. 19
Another straightforward assay for determining the amount of viable cells is to
measure radioactivity leakage from metabolically prelabeled cells after doping the
LYS