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248 Cell-Penetrating Peptides: Processes and Applications
b) Propidium
Iodide
a) Trypan Blue
N
c) 3 H-DG
3
H-DG
3
H-DG-P
FIGURE 11.1a Methods used for estimating CPP-induced membrane toxicity. (a) Membraneimpermeable
dyes (trypan blue, nigrosin, etc.) are excluded by living cells and can only enter
into dead cells with damaged membranes. (b) Fluorescent DNA intercalating dyes are impermeable
to cells and stain nuclei of damaged cells only. (c) Tritiated 2-deoxyglucose (DG) is taken
up into the cells by glucose carriers and phosphorylated. The resulting 3 H-2-deoxyglucose-
6-phosphate (DGP) efflux is only from cells with perforated membranes. (N = nucleus.)
instance by binding or cross-linking surface receptors. They may affect lipids by
disturbing lipid organization or by lipid peroxidation as a consequence of free radical
formation (e.g., ROS). Other agents act as detergents, dissolving membranes and
forming permeable pores. Through these pores protons, metal ions and even proteins
can leak out of the affected cell, causing collapse of the membrane potential and
eventually cell death.
Good correlation between increased plasma membrane permeability and cell
pathology enables rapid identification of dying or dead cells in vitro. 13 Exclusion of
impermeable dyes by viable cells and leakage of cytosolic proteins or introduced
markers from damaged cells are two ways to measure toxicity in vitro. 8,14
11.2.1.1.1 Exclusion of Dyes
Most cells are impermeable to colloidal dyes such as nigrosin, trypan blue, and
erythrosine B. 13,15,16 However, if the cell plasma membranes have been damaged or
if the cells have died, dye exclusion ability will be lost yielding colored cells
(Figure 11.1.a). The ability of cells to exclude the dye is so profound that it can be
applied for measuring cell viability. 15 Usually the cells are stained in suspension,
plated out, and viable cells counted using a hemocytometer. Several modifications,
such as spectrophotometric analysis for quantification by Saijo 17 and a perfusion
system for continuous reading of trypan blue uptake in cell monolayer cultures by
Walum and co-workers, 16 have been suggested in order to decrease the subjectivity
involved in this method. Similar to colloidal stains, impermeable fluorescent dyes
such as dansyl lysine are often used to identify damaged or dead cells. DNA
(a)