| Cell cytotoxicity as a measure of Natural Killer (NK) or
other immune cell effector function has traditionally
been examined using 51-Chromium release assays.1
Recently, to avoid costs associated with handling
and disposal of radioactive waste, a number of fluorescence
based flow cytometric assays have been
developed as alternatives.2–5 Here we describe a novel
assay in which the target cells, K562, are first labeled
or “painted” with CFSE (a fluorescent indicator dye),
incubated with NK effector cells and then monitored
for the percentage killed using a second fluorescent
indicator dye, 7-ADD. Both live and dead targets retain
the signal from the CFSE dye, as it is covalently linked
to intracellular amine groups upon internalization and
does not leach from cells into the media nor is transferred
to adjacent cells. Dead target cells in addition
take up the membrane impermeant 7-ADD dye. Thus,
on the benchtop microcytometry instruments—the
Guava PCA™ and PCA-96 systems—the live cells which
fluoresce orange can be readily distinguished from the
dead cells which fluoresce orange and red. Using this
assay, increasing amounts of dead target cells were
detected and quantified as increasing amounts of
NK effectors purified from whole blood were added.
Results were comparable to those obtained with other
NK assays. Increasing amounts of NK activity were also
detected when NK cells were pretreated with IL-2. The
Guava CellToxicity assaywas also used to demonstrate
potent T-cell mediated cytotoxicity (CMC) against
allogeneic target cells. Similar experiments can be
run on the same system for antibody-dependent
cell-mediated cytotoxicity (ADCC). Thus, the Guava
CellToxicity assay and the Guava platforms provide an
intuitive two-color system for assessing cytotoxicity in
a number of formats in which live and dead cells are
readily distinguished and analyzed based on their differential
fluorescence, and the data are displayed in a
convenient format for simple interpretation of percent
of killed target cells by the user. |
