Apoptosis is an evolutionarily conserved process of programmed cell suicide mediated by a cascade of proteolytic enzymes called caspases. Once activated in response to pro-apoptotic signals, caspases cleave protein substrates leading to the eventual disassembly of the cell.
Early research studies using short peptide sequences linked to an aldehyde or fluoromethyl ketone (FMK) reactive group identified a number of peptide sequences preferentially targeted by different caspase enzymes. When linked to an FMK reactive group, the resulting peptide-FMK molecule will form an irreversible, covalent thioether adduct with the reactive SH-site of caspase enzymes.
ImmunoChemistry Technologies utilized these findings to develop the often-cited FLICA® (FLuorescence Labeled Inhibitors of CAspases) caspase inhibitor and apoptosis detection reagents, both of which have been widely used for years as simple and reliable methods for screening apoptosis in live cells and tissues. These reagents consist of short peptide sequences with varying caspase specificities, a fluorescent label, such as carboxyfluorescein (FAM), and an FMK reactive moiety. The resulting fluorescent, cell-permeant, non-cytotoxic caspase inhibitor reagents can be added to cell culture media for whole cell apoptosis detection. These labeled caspase inhibitors will cross the cell membrane and form irreversible covalent bonds with activated caspase enzymes inside apoptotic cells, which can then be differentiated from non-apoptotic cells by their retained fluorescence.
ImmunoChemistry Technologies has released a novel set of inhibitor reagents that employ an O-phenoxy (OPH) reactive group in place of an FMK group. In a manner analogous to the FMK class of cysteine reactive compounds, the OPH caspase inhibitors form a stable, covalent thioether adduct with the reactive SH-site of caspase enzymes present in apoptotic cells. OPH inhibitor compounds have the benefit of being extremely non-cytotoxic, display enhanced stability characteristics compared to the FMK analogs, and have been optimized to provide a high level of performance for in vitro applications.
1. For in vitro apoptosis detection, culture your cells to a concentration of 2-5 x 105 cells/mL.
2. Prepare experimental and control populations; concentrate cells to 5 x 105-106 cells/mL in 300 µL samples.
3. Reconstitute the FAM-OPH reagent with 50 µL DMSO to form the 150X stock concentrate (may be frozen for future use).
4. Dilute the FAM-OPH stock concentrate with 200 µL PBS to form the 30X working solution.
5. Add FAM-OPH working solution directly to samples and controls at a ratio of 1:30. For example, add 10 µL of working solution to a 300 µL aliquot of suspension cells.
6. Incubate ~1 hour.
7. Wash and spin cells two or three times.
8. If desired, label cells with Hoechst 33342 nuclear stain, DAPI nuclear stain, or other compatible fluorescent markers.
9. If desired, fix cells with formalin-based agents.
10. Analyze data using a fluorescence microscope, plate reader, or flow cytometer.
Jurkat cells (T lymphocytes) were treated with DMSO as a negative control (left) or 1 μM staurosporine (right), an apoptosis inducer, for 3 hours at 37°C. FAM-VAD-OPH II Poly Caspase Inhibitor was added to the cell culture for 1 hour to detect active caspases, followed by a wash step. Cells were then dually-stained with the live-cell impermeant nuclear dye Propidium Iodide at 2.5μg/mL to detect membrane-permeant cells experiencing necrosis.
Cells were analyzed using two-color flow cytometry with a BD FACSCanto flow cytometer. Density plots were set up to detect caspase activity (green, FL-1) on the X-axis and necrosis (red, FL-2) on the Y-axis. Very few Jurkat cells were killed when treated with the placebo DMSO (left plot), while many cells entered apoptosis and secondary necrosis when treated with staurosporine (right plot).
Four populations of cells were detected:
1. Lower left: Non-fluorescent cells are live/ non-apoptotic;
2. Lower right: Cells fluorescing green from FAM-VAD-OPH II are in early apoptosis: caspase-positive but excluding PI nuclear vital dye with intact cell membranes;
3. Upper right: Dually-stained cells emitting green fluorescence from FAM-VAD-OPH II and red fluorescence from PI are in late apoptosis: caspase positive with permeabilized cell membranes;
4. Upper left: Cells emitting red fluorescence from PI are necrotic; no active caspases were present, and cell membranes were permeant to PI.
Data courtesy of Dr. Michael Olin, University of Minnesota (ICT-202:49; 072110.pptx).
• Target: apoptosis, poly caspases
• Final usage concentration: 10 µM
• Excitation/Emission: 488-492 nm / 515-535 nm
• Method of analysis: Fluorescence microscope, flow cytometer, fluorescence plate reader
• Types of samples: Cell culture, tissue
• Storage: 2-8°C
• Shipping: Ships overnight (domestic), International Priority Shipping