EMD Millipore eBook - 8

Analyzing Cell Death * So Many Ways to Die

of microcapillary flow cytometry, the Muse® is
an exceptionally compact and robust instrument
that capitalizes on optimized assays for crucial
cell death markers to dramatically simplify
the protocol for staining and acquisition of data
from every cell in a sample.
Simplified Flow Cytometric Determination
in Apoptosis and Autophagy
Early in the apoptotic process, molecules of
phosphatidylserine (PS) are translocated to the
outer surface of the cell membrane. The Muse®
Annexin V and Dead Cell Assay kit uses Annexin
V conjugated to the fluorophore phycoerythrin
(AnnexinV-PE) to bind and detect PS on the external membrane of apoptotic cells. Because it is
excluded from live and healthy cells, fluorescent
Figure 2. Data output for the Muse® MultiCaspase Assay. Jurkat cells were treated with staurosporine,
stained with the Muse® MultiCaspase Kit and analyzed on the Muse® Cell Analyzer. Summary data with
statistics (top) are produced automatically and include percentages and the concentration (cells/mL) for
the gated events in each quadrant, as well as the percentage and concentration of total caspase-positive
cells. Data is also displayed as dot plots (bottom). For each cell in the sample, caspase signal is plotted vs.
both cell size index (left) and vs. cell viability (right).
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signal from 7-AAD is used to differentiate live
from dead cells. By plotting these two parameters
on a scatter plot, the Muse software automatically
returns absolute numbers and percentages of four
distinct populations: live, dead, early apoptotic and
late apoptotic/dying cells.
Caspases (cysteinyl-directed aspartate-specific
proteases) are cysteine proteases that play a central
role in propagating the process of apoptosis in
response to external stress signals. In addition,
several caspases have also been found to have nonapoptotic roles in inflammation, mediating immunity,
cell fate specification, cell survival, cell cycle regulation,
cell proliferation, and cell migration. The Muse®
MultiCaspase kit is a pan-caspase assay that can
detect the presence of multiple caspases (caspase-1,
3, 4, 5, 6, 7, 8, and 9) (Figure 2).
The Muse® Caspase-3/7 Kit simultaneously
determines the count and percentage of cells in
various stages of apoptosis based on activity of socalled "executioner caspases" -3 and -7 as detected
by the novel caspase substrate reagent NucView, in
combination with the dead cell dye 7-AAD.

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