Green Fluorescent FAM-VAD-OPH II in vitro poly Caspase Apoptosis Detection Reagent
O-phenoxy (OPH) inhibitors form a stable covalent thioether adduct with the reactive -SH group of active caspase enzymes. FAM-VAD-OPH II poly caspase inhibitor can be used with a fluorescence microscope, fluorescent plate reader, or flow cytometer.
Figure 1. Jurkat cells were treated with DMSO (left) or staurosporine (right), labeled with FAM-VAD-OPH II, washed, and stained with PI. Four cell populations are seen: unstained live cells do not fluoresce (lower left); early apoptotic cells fluoresce green with FAM- VAD-OPH II (lower right); late apoptotic cells (upper right) fluoresce green (active caspases) and red (permeabilized cell membrane); and necrotic cells fluoresce red (upper left). Data of Dr. M. Olin, University of Minnesota.
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- For in vitro apoptosis detection, culture your cells to a concentration of 2-5 x 105 cells/mL.
- Prepare experimental and control populations; concentrate cells to 5 x 105-106 cells/mL in 300 µL samples.
- Reconstitute the FAM-OPH reagent with 50 µL DMSO to form the 150X stock concentrate (may be frozen for future use).
- Dilute the FAM-OPH stock concentrate with 200 µL PBS to form the 30X working solution.
- 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.
- Incubate ~1 hour.
- Wash and spin cells two or three times.
- If desired, label cells with Hoechst 33342 nuclear stain, DAPI nuclear stain, or other compatible fluorescent markers.
- If desired, fix cells with formalin-based agents.
- Analyze data using a fluorescence microscope, plate reader, or flow cytometer.