159molecule (M30) to which monoclonal antibodies have been raised
[ 11 ]. Unconjugated primary antibody targeting the neoepitope
can be used in western blotting but requires an additional second-
ary antibody conjugated with HRP (horseradish peroxidase). In fl ow
cytometry applications, the use of a fl uorochrome-conjugated anti-
cleaved CK18 antibody creates a powerful tool facilitating quanti-
fi cation of apoptotic cells in a culture. However, the use of CK18
neoepitope as an marker for apoptosis is restricted to epithelium-
derived cell lines. In non-epithelial cells, a similar protocol can be
used to target the cleaved fragment of PARP-1 [ 12 ].The function of a particular caspase in apoptotic cascades can be
determined by a strategy based on a series of peptide-based sub-
strates and inhibitors developed from the favored substrates of each
caspase. The caspase activity assay is a quantitative and sensitive
method which utilizes synthetic tetrapeptide or pentapeptide
sequences, conjugated with 7-amino-4-methylcoumarin (AMC),
7-amino-4-trifl uoromethylcoumarin (AFC), or rhodamine-110
(Rh110). When the low molecular weight substrate is cleaved by a
particular caspase, release of the fl uorescent molecule is detected
and the signal is proportional to the magnitude of the caspase pro-
teolytic activity. Alternatively, spectrophotometric detection of the
chromophore p-nitroanilide (p-NA) after cleavage from the labeled
substrate can be used. The p-NA light emission can be quantifi ed
using microtiter plate reader at 405 nm. However, it is important to
keep in mind that promiscuity for different cleavage motifs has been
reported. For instance, by testing commercially available short
peptide-based substrates in vitro and in cellulo, it was shown that
caspase-3 was able to cleave several substrates more effi ciently than
those caspases to which the substrates were reported specifi c [ 13 ].
Accordingly, the use of synthetic substrates to defi ne individual cas-
pases and discrete apoptotic signaling routes is controversial.In cells, the processing and activation of caspases is controlled by
apoptosis regulatory factors such as FADD (FAS-associated death
domain protein), APAF-1 (Apoptotic protease activating factor 1),
Bcl-2 family members, FLIP ( FLICE-like inhibitory protein ), and
IAPs (inhibitors of apoptosis proteins). Deregulation of these pro-
teins by means of overexpression using cDNAs or suppression by
RNAi methodology is in many experimental settings an effi cient
approach to manipulate apoptosis progression. Distinct from cas-
pase regulators produced by the cell itself, active caspases can also
be suppressed by artifi cial caspase inhibitors that have been devel-
oped both as research tools and as potential therapeutics to inhibit
cell death in vivo [ 14 ]. The simplest and therefore also the most
common method to accomplish inhibition of caspase activity in
cells is to culture them in the presence of micromolar concentra-
tions of cell-permeant and uncleavable substrate peptide motifs.1.5 Caspase Activity
Measurement
Using the Peptide
Cleavage Assay
1.6 Inhibition
of Caspase Activity
in Cell Cultures
Caspases in Mammalian Cell Cultures