CellEvent™ Caspase-3/7 Green ReadyProbes™ Reagent - FAQs

Yes, on both accounts. We have demonstrated that it works for microplates, and we have multiplexed the dye with PrestoBlue Cell Viability Reagent. Some microplates may be less sensitive than microscopic analysis, so sensitivity may not be the same for the CellEvent reagent.

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It has been done, but we don‘t recommend it. Both healthy cells and apoptotic cells possess phosphatidylserine on the cell surface, which can be detected with Annexin V, but apoptotic cells have significantly more of it. You can easily tell the difference between these two populations with flow cytometry, because flow cytometers are more sensitive and have a higher throughput. But with a microscope, you cannot always tell the difference, especially for adherent cells. Instead, for microscopy, we recommend a different technique, such as detecting caspases with CellEvent Caspase Detection Reagents.

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The recommended storage conditions for CellEvent Caspase-3/7 Green ReadyProbes Reagent (Cat. No. R37111) is between 4 and 25 degrees C or room temperature.

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We have tested Caspase 3/7 Green Detection Reagents (Cat. Nos. C10432, C10433, C10434, C10423, C10723, R37111) for up to 48 hours and observed no toxicity or stability issues with the reagent. If the samples are fixed at the end of the time course the signal will be retained for days. For live cells, apoptotic cells will round up and float away before the loss of fluorescence signal from CellEvent.

The following reference reports a visible signal for up to 72 hrs:

Sambi M, Samuel V, Qorri B, Haq S, Burov SV, Markvicheva E, Harless W, Szewczuk MR. A Triple Combination of Metformin, Acetylsalicylic Acid, and Oseltamivir Phosphate Impacts Tumour Spheroid Viability and Upends Chemoresistance in Triple-Negative Breast Cancer. Drug Des Devel Ther. 2020 May 25;14:1995-2019. doi: 10.2147/DDDT.S242514. PMID: 32546966; PMCID: PMC7260544.

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This is not recommended. The ReadyProbes reagents were developed for imaging applications whereas the Ready Flow reagents were optimized for flow cytometry.

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No, it must be applied to live cell populations.

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Yes, you may use SYTOX AADvanced Dead Cell Stain that is included in the CellEvent Caspase-3/7 Green Ready Flow Reagent (Cat. No. R37167) and in the CellEvent Caspase-3/7 Green Flow Cytometry Assay Kit (Cat. No. C10427, C10740). You may also try using other nucleic acid stains in the red or far red channels for staining cells.

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Yes. You can apply surface labels or other reagents. We recommend applying the surface labels or other reagents first.

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The CellEvent Caspase-3/7 Green Detection Reagent is not covalently attached to any cellular component and may be lost upon fixation. For flow cytometry applications, after applying the reagent, you should examine without washing or fixation. For imaging applications, the CellEvent product signal may be retained after fixation in 3.7% formaldehdye for 15 min.

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For imaging applications, we recommend fixing with 3.7% formaldehyde at room temperature for 15 mins. After fixation, cells can be imaged for up to 24 hrs, but the signal will gradually degrade over time. For flow cytometry applications, fixation is not recommended.

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It is possible to combine PrestoBlue reagent (for cell proliferation) with the CellEvent Caspase 3/7 Green Detection Reagent (for apoptosis detection) in the same sample.

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-Measures data from single cells.
-Data are obtained for a large number of cells, generating a rich statistical analysis of cell populations.
-Because single cells are measured, it will reveal heterogeneity within a population.
-With the ability to multiplex, small sub-populations can be identified.
-Thousands of cells can be analyzed rapidly.
-It is ideally suited for blood samples and other cells in suspension.
-Data can be re-analyzed multiple times after acquisition.
-Flow cytometry files (FCS) can be archived.

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There are several applications, some of which include immunophenotyping, cell cycle analysis, apoptosis assays such as annexin V staining, CellEvent Caspase-3/7 assay, and TUNEL assay, cell viability, proliferation assays such as CellTrace assay and Click-iT EdU assay, measurements of mitochondrial potential with MitoProbe assays, and cell counting using counting beads.

Find additional tips, troubleshooting help, and resources within our Flow Cytometry Support Center.