Figure 2. Dual detection of GFP and caspase-3/7 in EGFP-expressing BJAB cells using the CellEvent Caspase-3/7 Red Flow Cytometry Assay Reagent. BJAB cells expressing a gentamicin-resistant EGFP gene were treated with 100 µg/mL gentamicin for 24 hours to induce apoptosis. Samples were labeled using CellEvent Caspase-3/7 Red Flow Cytometry Assay Reagent and subsequently analyzed on an Attune NxT flow cytometer equipped with 488 nm and 637nm lasers. Fluorescence emission was collected using a 530/30 BP filter for EGFP detection and 670/14 BP filter for detection of CellEvent Caspase-3/7 Red. Cells not expressing gentamicin-resistant EGFP undergo apoptosis via a caspase-3/7-dependent mechanism.
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
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|
|CellEvent Caspase-3/7 Red Flow Cytometry Assay Reagent||3,7||633/635||630/650|
|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|
The CellEvent™ Caspase-3/7 Flow Cytometry Assays enable the flow cytometric detection of activated caspase-3 and caspase-7 in apoptotic cells using novel, fluorogenic caspase-3/7 detection reagents (Figures 1 and 2).
- 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
- Multicolor compatibility—available for 488 nm or 633 nm laser excitation
CellEvent Caspase-3/7 detection reagents are cell-permeant. Each 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 or 630/650 nm for the red detection reagent. When used together with the appropriate SYTOX dead cell stain, apoptotic cells can be easiliy 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.
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 and inhibits further enzymatic activity (Figure 3).
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 3. 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 4. 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.
For Research Use Only. Not for use in diagnostic procedures.