Invitrogen™ CellEvent™ Caspase-3/7 Detection Reagents

Yes, when the labels are spectrally and chemically compatible. For live-cell surface labeling, apply and optimize the surface label before adding the CellEvent Caspase reagent. For intracellular immunofluorescence, allow the CellEvent Caspase reagent signal to develop in living cells before fixation, permeabilization, and antibody staining. Use single-color controls to evaluate spectral overlap, background, and staining sequence effects.

Invitrogen CellEvent Caspase-labeled living cells can be fixed after signal development, but previously fixed cells cannot be stained for caspase-dependent signal. Add CellEvent Caspase Detection Reagents to living cells before fixation. After signal development, cells may be fixed with 4% paraformaldehyde for 20 min at room temperature and washed three times as one example. Fluorescence may remain detectable after fixation, but retention depends on fixation, washing, storage, and imaging conditions. Image fixed samples promptly or verify the required storage interval. Wash steps may remove loosely attached apoptotic cells.

Yes. Invitrogen CellEvent Caspase-3/7 Green Detection Reagent has been demonstrated in combination with Invitrogen PrestoBlue Cell Viability Reagent in the same sample. CellEvent Caspase reagent fluorescence reports activated caspase, while PrestoBlue Cell Viability Reagent reports cellular metabolic activity as an indicator of viability. Follow the instructions for each reagent and use single-reagent controls to evaluate signal interference.

Yes. A compatible fluorescence microplate reader can measure Invitrogen CellEvent Caspase reagent fluorescence as a population-level whole-well response. Optimize cell density, treatment conditions, assay timing, and reader settings, and include untreated and induced controls. Use imaging when single-cell localization or percentage-positive measurements are required.

Include untreated or vehicle-treated cells, an induced positive control, a no-reagent background control, and an inhibitor-pretreated condition when appropriate. The source protocol lists camptothecin with Z-DEVD-FMK for caspase-3/7 and staurosporine with Z-IETD-FMK or Z-LEHD-FMK for caspase-8 or caspase-9. Optimize inducer and inhibitor concentrations and timing for the cell model.

Quantify nuclear fluorescence using measurements such as percentage of caspase-positive cells, nuclear fluorescence intensity, and nuclear morphology. Use consistent acquisition settings and define positive thresholds relative to untreated controls.

CellEvent Caspase-3/7 Green, Caspase-8 Green, and Caspase-9 Green Detection Reagents, which have approximate excitation/emission maxima of 502/530 nm. Use a Texas Red dye optical configuration for CellEvent Caspase-3/7 Red Detection Reagent, which has approximate maxima of 590/610 nm.

Yes. The protocol can be adapted to additional cell types, but different cell models will require optimization. The user guide protocol was optimized using HeLa, A673, and U-2 OS cells. When evaluating a new cell model, titrate the reagent, verify induction conditions, and include positive, negative, and inhibitor controls.

Yes. Invitrogen CellEvent Caspase Detection Reagents may remain in complete medium during extended culture of planar 2D and 3D models. For spheroids, organoids, or other thick models, optimize reagent working concentration, treatment conditions, imaging depth, and analysis because geometry and diffusion can affect the measured response.

Yes. Use an environmentally controlled microscope or on-stage incubator maintained at the required culture conditions such as the Invitrogen EVOS M7000 Imaging System with the Invitrogen EVOS Onstage Incubator.

No. Invitrogen CellEvent Caspase Detection Reagents are designed for add-and-read use in complete culture medium or compatible physiological buffer. Medium composition and cell type can affect performance, so optimize reagent concentration and acquisition settings for new models. Avoiding a wash step may also reduce loss of loosely attached apoptotic or dying cells.

Signal timing depends mainly on when the target caspase becomes active. For endpoint measurements, allow 30-60 minutes at 37°C after adding the reagent before measurement.

Start at 5 µM for Invitrogen CellEvent Caspase-3/7 Green, Caspase-3/7 Red, Caspase-8 Green, and Caspase-9 Green Detection Reagents; optimize within 2-10 µM for a new model. For CellEvent Caspase-3/7 Green ReadyProbes Reagent, add two 40 µL drops per mL of complete medium.

Add a freshly prepared 10X Invitrogen CellEvent Caspase staining solution to living cells at a 1:10 dilution in complete culture medium or a compatible physiological buffer. Treat the cells with the selected apoptosis inducer as required, incubate with the reagent for 30-60 minutes at 37°C, and measure fluorescence. Cell lysis, permeabilization, and washing are not required before measurement.

Yes, but use matched parallel wells for Invitrogen CellEvent Caspase-3/7 Green, Caspase-8 Green, and Caspase-9 Green Detection Reagents since their spectra overlap, so they are not distinguishable in the same fluorescence channel when combined. CellEvent Caspase-3/7 Red Detection Reagent may be paired with either green reagent after experimental verification with single-color controls.

Caspase responses can vary because cell type, culture medium, cell density, treatment, pathway composition, and activation timing affect staining and signal. Evaluate new cell models with untreated, induced, and inhibitor-treated controls.

Choose Annexin V to detect phosphatidylserine exposure at the plasma membrane, and choose Invitrogen CellEvent Caspase Detection Reagents to detect activated caspases and nuclear fluorescence. The methods measure different apoptotic events and should not be treated as interchangeable.

Choose Invitrogen CellEvent Caspase Detection Reagents when you need live-cell monitoring, kinetic measurements, image-based cellular context, or signal that can be retained after fixation. Luminescence-based caspase assays commonly lyse cells and generate a population-level well signal for plate-based endpoint quantitation. Invitrogen CellEvent Caspase assays can also be measured with compatible fluorescence microplate readers.

Invitrogen CellEvent Caspase reagents are cleavable substrates, whereas FLICA reagents are fluorescent caspase inhibitors that covalently bind active caspases. FLICA workflows typically require removal of unbound reagent, and the bound reagent inhibits the labeled enzyme. CellEvent Caspase reagents generate nuclear fluorescence without inhibiting caspase activity and do not require a wash step.

Interpret the signals as measurements of caspase activity, not definitive evidence of a single apoptosis pathway. Pathway crosstalk, feedback, stimulus, cell type, and activation timing can affect which caspases become active. Compare matched cell density, treatment conditions, acquisition settings, and time points when evaluating activation patterns.

Choose according to the caspase target and fluorescence channel. Use Invitrogen CellEvent Caspase-3/7 Green or Red Detection Reagent for downstream executioner caspase activity, Invitrogen CellEvent Caspase-8 Green Detection Reagent for initiator activity generally associated with death-receptor signaling, and Invitrogen CellEvent Caspase-9 Green Detection Reagent for initiator activity generally associated with mitochondrial apoptosome signaling. Choose the red Caspase-3/7 reagent when GFP or another green fluorescent marker is present.

Each Invitrogen CellEvent Caspase Detection Reagent contains a caspase-recognition peptide linked to a proprietary fluorogenic DNA-binding dye. Caspase cleavage releases the dye so it can bind nuclear DNA and fluoresce. CellEvent Caspase-3/7 reagents use the DEVD recognition sequence, CellEvent Caspase-8 Green Detection Reagent uses IETD, and CellEvent Caspase-9 Green Detection Reagent uses LEHD.

Invitrogen CellEvent Caspase Detection Reagents are cell-permeant, fluorogenic substrates used to detect activated caspase-3/7, caspase-8, or caspase-9 in living cells. They generate nuclear fluorescence in caspase-positive cells and support endpoint, kinetic, continuous-culture, high-content imaging, and fluorescence microplate reader workflows.