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- Prepared samples can be stored in methanol at −20 °C for
several weeks. - To prevent clustering of cells which could interfere with fl ow
cytometry analysis, ice cold methanol should be added drop
wise to cells while wortexing. - For statistical calculations triplicate samples for each condition
are recommended. - The results obtained have to be recalculated with respect to
input of total protein amount, or the number of cells present
in individual samples. Since this is critical in order to accom-
plish accurate assay readout, it is recommended to do both. - The freezing of samples is performed in order to permeabilize
plasma membranes. If cells are lysed by a protocol which main-
tains protein function, cell lysates can equally well be used as
templates for the caspase activity assay. Frozen samples can be
stored for several days at −20 °C. - Caspase inhibitors are functionally stable in cell cultures at
least 48 h. - Apoptosis in cells can be monitored by a phase-contrast micro-
scope. Attached cell types are normally detached as a result of
apoptosis. The dramatic change of morphology, due to plasma
membrane blebbing and cell shrinkage, can be used to observe
apoptotic conditions in suspension cells.
5 Conclusions
Since detection of caspase activities is one of the most common
biochemical markers of apoptosis, several techniques have been
developed to characterize this process. Although most of them are
provided by various companies as method kits, comparable results
can certainly be obtained by purchasing components and reagents
separately and then, take advantage of shared protocols. Caspases
are activated in a signaling cascade where a specifi c effector caspase
is processed by one or several types of apical caspases. In turn, effec-
tor caspases may further activate apical caspases as part of an apop-
tosis amplifying loop. Thus, caspases are generally activated jointly.
Once activated, caspases cleave target substrates with some degree
of overlapping specifi city. Consequently, all assays described in this
chapter are appropriate for the detection of apoptosis per se. In
order to make further conclusions regarding the role of individual
caspases in a given system, it is highly recommended to also suppress
the caspase of interest by additional techniques, such as. Another
aspect that should be considered in advance of experimental design
is timing. Initiation of cell death signaling may occur in minutes or
days depending on the compound or culture condition used forMagnus Olsson and Boris Zhivotovskyhttp://www.ebook3000.com