Measurement of CTL- and NK-mediated target cell cytolysis by flow or image cytometric detection of caspase 6 activity.
Elena Kovalenko1, Dan Fowler2, Karen Abdool2 and Bill Telford2
1Laboratory of Cell Interactions, Division of Immunology, Shemyakin and Ovchinnikov Institute of Bioorganic Chemistry, Moscow, Russia; 2Experimental Transplantation and Immunology Branch, NCI-NIH
NK- and CTL-mediated cytolysis of target cells constitutes a form of apoptosis, and is accompanied by many of the same events associated with receptor-mediated apoptosis, including caspase activation. OncoImmunin, Inc. (Gaithersburg, MD) has developed a fluorogenic substrate for caspase 6 (similar to their PhiPhiLux G1D2 substrate specific for caspase 3) that can be incorporated into a target cell line and can measure caspase activation following coincubation with cytolytic cells. The kit (termed CyToxiLux) allows prior tagging of the target cell line with an proprietary orange tracking dye followed by substrate loading and plating with the cytolytic cell of interest. The cells are then analyzed by single-laser flow cytometry, gating for tagging dye-positive cells. This assay can also be adapted to laser scanning cytometry (below).
Protocol for NK-mediated cytolysis analysis by caspase 6 detection. NK92
cells were grown in modified Iscoves medium supplemented with 10% FBS and 10%
Myelocult. Human NK cells were isolated from RBC-depleted human blood using the
Miltenyi Biotec negative selection magnetic bead system. K562 target cells were
labeled with the proprietary tracking dye included in the CyToxiLux kit and coincubated
with NK effectors at the indicated ratios for 1 hour. Cells were subsequently loaded
with CyToxiLux caspase 6 fluorogenic substrate for 45 minutes at 37C, washed and analyzed
on a Becton Dickinson FACScan equipped with a 488 nm air-cooled argon laser. Caspase
6 substrate fluorescence was measured through the FL1 channel (530/30 nm filter) and the
tracking dye through the FL2 channel (575/26 nm filter). Data is displayed for
caspase 6 fluorescence following gating for the tracking dye. Detailed CyToxiLux
protocol 
here.
(Below). Flow chart for CyToxiLux assay.
(Below). NK92- and human NK-mediated K562 cytolysis. NK92 or human NK cells isolated as described above were coincubated for 1 hour with tracking dye labeled K562 cells at the indicated ratios and subsequently loaded with fluorogenic caspase 6 substrate. Percentage positive cytotoxicity is shown for each K:T ratio.
Data courtesy of Dr. Elena Kovalenko, Shemykin and Ovchinnikov Institute of Bioorganic Chemistry.
Protocol for CTL-mediated cytolysis analysis by caspase 6 detection in adherent target cells. Adherent TSA epithelial breast carcinoma cells were plated onto 8-well tissue culture coated glass culture slides for 24 hours at 20,000 cells/200 ul/well to ensure good cell distributions. CD8-positive effector T cell clones originally derived from B6 transgenic mice Tc1 and Tc2 lines shown here) were activated with human IL-2 for 48 hours prior to the assay and plated over the target layers for three hours. Effector cells were then removed by multiple washes and the monolayers loaded with fluorogenic caspase 6 substrate at 2 uM/100 ul concentration for 1 hour, followed by one wash with complete media and subsaequent incubation with 7-AAD at 5 ug/ml in complete media for 15 minutes. Slides were coverslipped and analyzed on the LSC for scatter, caspase 6 and 7-AAD fluorescence. Data was displayed caspase 6 activity gated for either all or 7-AAD-negative cells. As in previous studies with LSC, max pixel analysis of caspase fluorescence proved to be more sensitive than integral fluorescence measurement.
(Below). Flow chart for CTL-mediated cytolysis analysis by caspase 6 detection in adherent target cells.
(Below). CTL-mediated cytolysis analysis by caspase 6 detection in adherent target cells. TSA target cells were refractory to cytolysis by Tc1 effectors (top four panels) but sensitive to Tc2 (bottom four panels). Slide well X-Y positions are shown in the left-most panels, 7-AAD versus caspase 6 activity is shown in the second panel and histogram. Max pixel caspase 6 fluorescence for ungated and 7-AAD-negative gated cells are shown in the third and right-most histograms.
(Below). Comparison of max pixel and integral fluorescence measurement of caspase 6 activity.
Download Adobe Acrobat Reader here .
For more information about Oncoimmunin's CyToxiLux reagent, click here.
The NCI ETIB Flow Cytometry Core laboratory has pilot quantities of these reagents available for development work by ETI Branch faculty. Talk to us for details.