CellEvent® Caspase-3/7 Green ReadyProbes™ Reagent is a fluorogenic, no-wash indicator of activated caspase-3/7 for live- and fixed-cell applications. Activation of caspase-3 is an early indicator of apoptosis and CellEvent® Caspase-3/7 Green reagent allows rapid and sensitive detection of cells destined for cell death. Like other ReadyProbes™ reagents, CellEvent™ Caspase-3/7 Green reagent is formulated as a room temperature-stable solution and comes in a convenient dropper bottle. Just tip and drip two drops per ml to stain your cells.CellEvent™ Caspase-3/7 Green ReadyProbes™ Reagent provides:
• Optimized caspase-3/7 substrate for apoptosis analysis
• Simple and fast no-wash protocol helps preserve delicate apoptotic cells
• Compatible with both live-cell fluorescence imaging and formaldehyde-based fixation methods
• Ready-to-use liquid formulation in convenient dropper bottle—no need to dilute, weigh, or pipette
• Stability at room temperature—keep handy at your scope or cell culture areaPrinciples of CellEvent® Caspase-3/7 Apoptosis Detection
CellEvent® Caspase-3/7 Green reagent is a four amino acid peptide (DEVD) conjugated to a nucleic acid-binding dye that is non-fluorescent when not bound to DNA. The CellEvent® Caspase-3/7 Green reagent is intrinsically non-fluorescent, as the DEVD peptide inhibits binding of the dye to DNA. Upon activation of caspase-3/7 in apoptotic cells, the DEVD peptide is cleaved and the free dye can bind DNA, generating a bright green fluorescence. The fluorescence emission of the dye when bound to DNA is 530 nm and can be observed using a standard FITC filter set. Simple Three-step Protocol
To use CellEvent® Caspase 3/7 Green ReadyProbes™ Reagent, simply add 2 drops/mL to cells, incubate 30 minutes, and visualize (Figure 1). Apoptotic cells with activated caspase-3/7 will have bright green nuclei, while cells without activated caspase 3/7 will show minimal fluorescence (Figure 2). Assay Versatility Allows for Live Cell Detection and Fixation
Wash steps are not necessary for detection, so fragile apoptotic cells are retained. CellEvent® Caspase 3/7 Green ReadyProbes™ reagent tshows excellent accuracy and robustness. Importantly, the fluorescent signal from CellEvent® Caspase 3/7 Green reagent survives fixation and permeabilization, allowing end-point assays and immunocytochemistry-based methods. Packaged for Convenience
CellEvent™ Caspase-3/7 Green ReadyProbes™ reagent is supplied ready-to-use and can be stored right next to your microscope or workstation. A liquid formulation in an ultra convenient dropper bottle (Figure 3) allows you to stain your cells whenever and wherever they are ready. Just add two drops per ml for optimally bright and distinct nuclear counter-staining. Suggestions for Use
• CellEvent™ Caspase-3/7 Green reagent may be added directly to fixed cells in full media or buffer solutions.
• In most cases, 2 drops/ml and an incubation time of 30 minutes is sufficient for bright nuclear staining of apoptotic cells; however, optimization may be needed for some cell types, conditions, and applications. In such cases, simply add more or fewer drops until the optimal staining intensity is obtained.
• If desired, combine CellEvent™ Caspase-3/7 Green ReadyProbes™ reagent with a red or far-red nuclear stain for a total cell count
• CellEvent™ Caspase-3/7 Green dye is excited with a maximum at 502 nm and has an emission maximum at 530 nm. It is detected through standard GFP and FITC filters. See other Molecular Probes® imaging tools and reagents