Green Fluorescent FAM-VAD-OPH II in vitro poly Caspase Apoptosis Detection Reagent

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.

Apoptosis is an evolutionarily conserved process of programmed cell suicide that centers on a cascade of proteolytic enzymes called caspases that are triggered in response to pro-apoptotic signals. Once activated, 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. For example, the sequence Asp-Glu-Val-Asp (DEVD) is cleaved by caspase-3 and caspase-7, the sequence Val- Ala-Asp (VAD) demonstrates multiple caspase (caspase -1, -2, -3, -4, -5, -6, -7 -8, -9) recognition properties. ImmunoChemistry Technologies utilized these findings to develop the often-cited FLICA® (Fluorescence Labeled Inhibitors of CAspases) caspase inhibitor reagents, which have been widely used for years as simple and reliable methods for screening apoptosis in live cells and tissues. These reagents were made by sandwiching target peptide sequences with varying caspase specificities between a green fluorescent label, carboxyfluorescein (FAM), and an FMK reactive moiety. The resulting green fluorescent, cell-permeant, non-cytotoxic inhibitor reagents can be added to cell culture media for in vitro use. They will cross the cell membrane and form irreversible covalent bonds with activated caspase enzymes present inside apoptotic cells, which can then be differentiated from non-apoptotic cells by their retained green fluorescence. ImmunoChemistry Technologies has now released a novel set of inhibitor reagents that employ an O-phenoxy (OPH) reactive group instead of an FMK group. In a manner analogous to the FMK class of cysteine reactive compounds, the OPH 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.