Fluorescent assays for the detection of apoptosis-associated caspase activity.
Caspase activation is a cellular event associated with the onset of apoptotic death. Detection of caspase activity provides a useful assay for analyzing one of the earliest known biochemical events associated with apoptosis. A variety of fluorogenic caspase substrates have been developed for the detection of functional caspase activity. Several such methods are described here; one used the PhiPhiLux caspase 3 substrate developed by OncoImmunin, Inc., for both flow cytometry and laser scanning cytometry, and and another a monoclonal antibody against the active form of caspase 3. Both techniques have been used to detect caspase activation and early cell death onset in intact cells by flow cytometry.
Measurement of caspase 3 activity in murine EL4 cells using the PhiPhiLux fluorogenic caspase 3 substrate.
OncoImmunin, Inc. (Gaithersburg, MD) has developed a unique set of enzyme substrates using two fluorochrome molecules coupled to either end of a substrate peptide. The fluorochromes are coupled in such a way as to maintain the molecules in close physical proximity; as a result, the intact complex is fluorescently quenched and shows relatively little fluorescent emission upon excitation. When the bridging peptide is cleaved, however, the fluorochromes are released and become fluorescent. A caspase 3 substrate complex of this type is available (termed PhiPhiLux) and is cell-permeable, allowing it to be loaded into cells for the intracellular detection of caspase 3. We have used both the G2D2 and G1D2 forms of the reagent (distinguished by the coupled fluorochrome structure and spectral characteristics). PhiPhiLux G1D2 can be excited at 488 nm and detected through a FITC filter; hence, it is the most applicable for flow and image cytometry.
(Below). Detection of cell death with PhiPhiLux G1D2. Murine EL4 cells were either harvested in log phase (let panel), treated with the DNA polymerase inhibitor quercetin (second panel from left) or the translation blocker actinomycin D (third panel from left) or deprived of serum (right panel) for 16 hours. Cells were then labeled with PhiPhiLux G1D2 according to the manufacturers directions and analyzed on a FACSVantage SE using argon-ion 488 nm excitation and a standard FITC filter (530/30) for detection. The caspase 3-positive subpopulation is generally one to two ordrs of magnitude brighter than the caspase-negative fraction. The intact complex possesses a small amount of fluorescence compared to background (unfilled peak in the viable histogram).
Apoptotic cells undergo morphological changes and can often be distinguished from "viable" cells by their reduced forward scatter and increased side scatter signals. Measurement of caspase 3 activation in these subpopulations nevertheless detects a fraction of caspase 3-active cells in the morphologically "viable" fraction. Caspase 3 activation therefore precedes the morphological changes in apoptotic death; a fraction of morphologically "viable" cells may therefore be preapoptotic. This distinction is important in measuring surface marker expression in a population of supposedly "viable" cells.
(Below). Relationship between caspase 3 activation and changes in cell morphology. "Viable" and apoptotic EL4 cells can be distinguished based on their scatter properties, the apoptotic cells showing reduced forward scatter and increased side scatter. Measurement of caspase 3 activity with PhiPhiLux G1D2 in both populations shows a caspase 3-high cell populaton in the supposedly "viable" cell fraction based on side scatter. These cells are presumably in the earliest stages of apoptosis and have not yet undergone the morphological changes associated with cell death.
Since the PhiPhiLux system can be used to detect functional caspase 3 activity in intact cells, it can be combined with other cell death assays more fully define the order of apoptotic events (such as annexin V binding to PS moieties and permeability to DNA binding dyes such as PI and 7-aminoactinomycin D). The PhiPhiLux system can also be combined with fluorescent immunophenotyping, allowing the measurement surface marker expression in the truly viable subpopulation (characterized as caspase 3-negative). Since some morphologically "viable" cells show elevated caspase levels (as illustrated above), this allows these preapoptotic cells to be separated from the viable fraction for measurement of surface marker expression.
(Below). Simultaneous analysis of annexin V binding, 7-AAD permeability and caspase 3 activation. EL4 Cells with either no additions or actinomycin D treatment for 8 hours were incubated with PhiPhiLux G1D2 for 1 hour at 37C, APC-annexin V for 15 minutes at room temperature, washed and subsequently incubated with 7-AAD at 5 ug/ml to 10 minutes prior to analysis. Cells were then analyzed for annexin V binding and 7-AAD incorporation - the histograms at left show PhiPhiLux fluorescence for ungated or annexin V-negative 7-AAD-negative.
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Measurement of caspase 3 activity in adherent mouse L929 cells by laser scanning cytometry.
Laser scanning cytometry is the best method for analyzing apoptosis in adherent cell lines. Removal of cells from their substrate for traditional flow cytometry can disrupt them sufficiently to alter their apoptotic characteristics. For example, cell scraping and trypsinization can cause aberrant flipping of phosphotidylserine residues to the outside membrane leaflet, causing viable cells to appear apoptotic. Cell removal can also also disrupt PhiPhiLux detection of caspase activation. Labeling and analysis of apoptotic cells in the adherent state using laser scanning cytometry preserves their apoptotic phenotype. One caveat - late apoptotic cells tend to "round up" and release from their substrate, possibly reducing the apparent level of apoptosis over analysis of suspension cells. However, 7-AAD-positive L929 cells are still detectable in the experiment below, suggesting that cel release is a relatively late apoptotic phenomenon.
(Below). Detection of apoptosis in L929 cells with 7-AAD and PhiPhiLux caspase substrate. L929 cells were induced to undergo apoptosis with TNF-alpha and cycloheximide, labeled with PhiPhiLux and 7-AAD and analyzed by laser scanning cytometry. Left image is an epifluorescence micrograph of caspase 3 activation. The upper left cytogram shows 7-AAD versus PhiPhiLux fluorescence, with a 7-AAD cytogram for all cells in the upper right. Lower left and right histograms show caspase 3 activity for all and 7-AAD-negative gated cells respectively.
Click here to download our methods paper for LSC detection of apoptotic cells...
Measurment of caspase 3 activity using a monoclonal antibody against active caspase.
Pharmingen has developed a monoclonal antibody against the cleaved, active form of caspase 3 (this antibody should not be confused with the earlier polyclonal version also available from Pharmingen, which has higher background levels). This antibody is reportedly specific for the active form of caspase 3, with significantly reduced affinity for the pro- form of the enzyme. Cells can be fixed and permeablized with 2% paraformaldhyde and 0.05% Tween 20 to allow intracellular labeling with this reagent. It has been successfully used by us to detect caspase 3 activation in both human (A1.1 and Jurkat) and mouse cells (EL4 thymoma and mouse thymocytes).
(Below). Detection of apoptosis using monoclonal antibody against the active form of caspase 3. EL4 cells were incubated with either no additions or actinomycin D, fixed with 1% paraformaldehyde in PBS containing 0.05% Tween 20 and labeled with either FITC- or PE-conjugated anti-active caspase 3. Actinomycin D panel shows caspase 3 histograms for b oth ungated and scatter-viable gated cells.
Cells can be sorted for functional caspase 3 activity based on PhiPhiLux, fixed and labeled with anti-caspase 3 antibody to determine the correlation between PhiPhiLux cleavage and active caspase 3 immunolabeling. Sorted functional caspase 3-positive cells all label with the antibody, suggesting good correlation between substrate cleavage and immunodetection.
(Below). PhiPhiLux caspase 3-positive cells are also positive for cleaved caspase 3 based on immunolabeling. EL4 cells were incubated with actinomycin D, labeled with PhiPhiLux G1D2 and sorted into scatter- viable PhiPhiLux-negative and -positive subfractions. These fractions were then fixed and labeled with PE-anti-active caspase 3. PhiPhiLux showed excellent correlation with anti-active caspase 3 labeling.
Click here for BD Pharmingen's data sheet
for anti-active caspase 3.
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.