CellEvent™ Caspase-3/7 Detection Reagents

CellEvent Caspase-3/7 Green and CellEvent Caspase-3/7 Red detection reagents are novel fluorogenic substrates for activated caspase-3/7 in live cells that can be fixed and multiplexed with antibody detection workflows such as immunofluorescence (IF) and immunocytochemistry (ICC). Activation of caspase-3 is an essential event during apoptosis, making these optimized reagents for analysis of apoptotic cells. CellEvent Caspase-3/7 Red Detection Reagent can also be used to detect apoptosis in GFP expressing cells.

Features of these indicators include:
• Optimized caspase-3/7 substrate for apoptosis analysis
• Easy to use—add and read; no cell lysis required
• Use for time course measurements; easily select the time point of interest
• Compatible with both live cell fluorescence-imaging and formaldehyde-based fixation methods
• Multiplex enabled—combine with other fluorescent reagents to confirm apoptosis in the same cell or cell population

CellEvent™ Caspase-3/7 Green Detection Reagent: Absorption/emission maxima of ∼502/530 nm. Detectable with FITC/GFP traditional filter set.

CellEvent™ Caspase-3/7 Red Detection Reagent: Absorption/emission maxima of ∼590/610 nm. Detectable with traditional Texas red filter set.

CellEvent™ Caspase-3/7 Red Detection Reagent and CellEvent™ Caspase-3/7 Green is provided as Dry down powder. Just add 100μL of PBS to dry down powder to generate 100X Stock solution. Dilute this 100X stock solution 1:100 in complete media and stain cells for 30-60 minutes and analyze with fluorescence microscope.

CellEvent™ Caspase-3/7 Red Detection Reagent is also provided as already prepared DMSO solution. DMSO solution is 400X stock solution, which is ready to use. Just dilute in growth media 1:400 and stain the cells for 30 to 60 minutes.

To prevent the apoptotic cells from washing away, no wash is needed at the end of staning. Stained cells can be analyzed live, or can be fixed with 4% Paraformaldehyde solution such as Image-IT™ Paraformaldehyde 4% in 0.1M Phosphate Buffer pH 7.4, Cat. No. I28800. These fixed cells can be further processed for ICC workflow.

Substrate specifics
CellEvent Caspase-3/7 Detection Reagents are a four-amino acid peptide (DEVD) conjugated to a nucleic acid-binding dye. The DEVD peptide sequence is a cleavage site for caspase-3/7, and the conjugated dye is non-fluorescent until cleaved from the peptide and bound to DNA.

Principles of action
CellEvent Caspase-3/7 Green Detection Reagent is intrinsically non-fluorescent as the DEVD peptide inhibits the ability of the dye to bind to DNA. However, after activation of caspase-3/7 in apoptotic cells, the DEVD peptide is cleaved, enabling the dye to bind to DNA and produce a bright, fluorogenic response.

Simple three-step protocol
To use CellEvent Caspase 3/7 Detection Reagent, simply add it to cells, incubate 30–60 minutes, and visualize. Apoptotic cells with activated caspase-3/7 will have bright green or red nuclei, while cells without activated caspase 3/7 will have minimal fluorescent signal.

Assay versatility allows for live cell detection and fixation
This robust assay enables the examination of caspase-3/7 activation in live cells. Furthermore, since wash steps are not necessary for detection, fragile apoptotic cells typically lost during wash steps are preserved. Importantly, the fluorescent signal from CellEvent Caspase 3/7 Detection Reagent can survive fixation and permeabilization. This allows for the flexibility to perform end-point assays and probe for other proteins of interest using immunocytochemistry.