A distinctive feature of the early stages of apoptosis is the activation of caspase enzymes, which participate in the cleavage of protein substrates and in the subsequent disassembly of the cell. We provide a series of caspase assays that allow the simple detection of active caspases in living cells by flow cytometry.
See also caspase assays for imaging
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Selection guide—caspase assays for flow cytometry
|Product||Caspase detected||Laser (nm)||Ex/Em (nm)|
|CellEvent Caspase-3/7 Green Flow Cytometry Assay Kit||3,7||488||511/533|
|Vybrant FAM Poly Caspases Assay Kit||All||488||495/529|
|Vybrant FAM Caspase-3 and -7 Assay Kit||3,7||488||495/529|
|Vybrant FAM Caspase-8 Assay Kit||8||488||495/529|
Real-time monitoring with the no-wash CellEvent Caspase-3/7 assay
The CellEvent Caspase-3/7 Flow Cytometry Assay enables the flow cytometric detection of activated caspase-3 and caspase-7 in apoptotic cells using novel, fluorogenic caspase-3/7 detection reagents (Figure 1).
- Caspase-3/7 specific—includes the recognition sequence for activated caspase-3 and caspase-7
- Easy identification—can be used to clearly identify live, dead, and apoptotic cell populations
- Quick analysis—washing and fixation is not required
- Compatibility—available for 488 nm excitation
CellEvent Caspase-3/7 detection reagent is cell-permeant. The reagent consists of a four-amino acid peptide (DEVD) conjugated to a nucleic acid-binding dye. During apoptosis, caspase-3 and caspase-7 proteins are activated and able to cleave the caspase 3/7 recognition sequence encoded in the DEVD peptide. Cleavage of the recognition sequence and binding of DNA by the reagent labels the apoptotic cells with a bright, fluorogenic signal that has absorption/emission maxima of ~511/533 nm for the green detection reagent. When used together with the appropriate SYTOX dead cell stain, apoptotic cells can be easily discriminated from live and necrotic cells.
Figure 1. Caspase activity detection in Jurkat cells using the CellEvent Caspase-3/7 Green Flow Cytometry Assay Kit on the Attune Flow Cytometer. Jurkat cells (T-cell leukemia, human) were treated with (A) DMSO or (B) 10 µM camptothecin for 3 hours before labeling with the CellEvent Caspase 3/7 Green Flow Cytometry kit. Stained samples were analyzed on the Attune Acoustic Focusing Cytometer equipped with a 488-nm laser, and fluorescence emission was collected using a 530/30 BP filter for CellEvent Caspase 3/7 Green Detection Reagent and a 690/50BP filter for SYTOX AADvanced stain, respectively. Note that the treated cells have a higher percentage of apoptotic cells (panel B) than the basal level of apoptosis seen in the control cells (panel A). A = apoptotic cells, V = viable cells, N = necrotic cells.
End point assay using fluorescent inhibitors of caspases
The Vybrant FAM Caspase Assay Kits employ a novel approach to detect active caspases in living cells, based on fluorescent inhibitor of caspases (FLICA) methodology. Vybrant FAM Caspase Assay Kits employ a novel caspase inhibitor, FLICA reagent, which binds covalently and irreversibly to the reactive cysteines of active caspases. This binding inhibits further enzymatic activity (Figure 2) and makes this an end point assay for caspase activity (Figure 2).
The assays are simple to use, and there's no need for special buffers to lyse or permeabilize the cell membranes—just add the reagent to your cell culture media, let it incubate 1–4 hours, wash the cells, and analyze.
- Allows for accurate detection of active caspases in living cells
- More reliable than annexin V
- Simpler than TUNEL assays
Figure 2. Detecting active caspases in apoptotic cells using the Vybrant FAM Caspase Assay Kits. Unbound FLICA reagent diffuses out of the cell and is washed away. The green-fluorescent signal is a direct measure of the amount of active caspase that was present at the time the inhibitor was added.
Figure 3. Detection of active caspases in living cells by flow cytometry. Jurkat cells were either treated with 10 μM camptothecin for 4 hours at 37ºC and 5% CO2 and stained with the FLICA reagent for caspase-3 and -7 and propidium iodide, both from the Vybrant FAM Caspase-3 and -7 Assay Kit. Samples were analyzed on a flow cytometer with 488 nm excitation using 530 nm bandpass and 670 nm longpass emission filters.
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