Caspase Assays for Flow Cytometry

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.  Life Technologies™ provides a series of caspase assays that allow the simple detection of active caspases in living cells by flow cytometry.

See All Caspase Assays

CellEvent® Caspase-3/7 Green Flow Cytometry Assay Kit

The CellEvent® Caspase-3/7 Green Flow Cytometry Assay Kit enables the flow cytometric detection of activated caspase-3 and caspase-7 in apoptotic cells. The kit includes the novel fluorogenic substrate CellEvent® Caspase-3/7 Green Detection Reagent, as well as SYTOX® AADvanced™ Dead Cell Stain.

  • Caspase-3/7 specific—includes the recognition sequence for activated caspase-3 and caspase-7
  • Easy identification—clearly identify live, dead, and apoptotic cell populations
  • Quick analysis—washing and fixation is not required
  • Multicolor compatibility—combine with other dyes excitable by the 488 nm laser or other lasers

CellEvent® Caspase-3/7 Green Detection Reagent is a cell-permeant reagent that 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. When used together with the SYTOX® AADvanced™ Dead Cell Stain, apoptotic cells can be easiliy discriminated from live and necrotic cells.

 

 

CellEvent® Caspase-3/7 Green Flow Cytometry Assay Kit

 
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.

 

Product Laser Ex/Em Cat. No.  
CellEvent® Caspase-3/7 Green Flow Cytometry Assay Kit488511/533C10427product detail

Vybrant ® FAM™ Caspase Assay Kits

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 1).

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 1. 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 2. 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.

 

Conjugate Caspase
Detected
Laser Ex/Em Cat. No.  
Vybrant® FAM™ Poly caspases assay kitAll488495/529V35117product detail
Vybrant® FAM™ caspase-3 & -7 assay kit3,7488495/529V35118product detail
Vybrant® FAM™ caspase-8 assay kit8488495/529V35119product detail

PARP Proteolysis

During apoptosis, ICE family members such as caspase-3 and -7 cleave PARP to yield an 85 kDa and a 25 kDa fragment. PARP cleavage is considered to be one of the classical characteristics of apoptosis. This FITC-conjugated anti-PARP antibody specifically recognizes the 85 kDa fragment of cleaved PARP and can be used as a marker for detecting apoptotic cells. The Anti-PARP FITC Apoptosis kit contains all the components and simplified protocol necessary to analyze PARP cleavage using flow cytometry.

 


Figure 3. Detection of apoptotic HeLa cells (adherent) by flow cytometry using FITC-PARP cleavage site–specific antibody. HeLa cells were treated with 0.5 μM staurosporine for 5 hours at 37ºC (low panels) and uninduced HeLa cells were used as control (upper panels). The cells were stained with 10 μL FITC-conjugated anti-PARP CSSA for 30 minutes and analyzed with FACScan. The data show that PARP CSSA stained apoptotic HeLa cells treated with staurosporine (lower left) but not uninduced HeLa cells (upper left). The positive staining in apoptotic cells can be blocked with the synthetic peptide corresponding to the N-terminus of cleavage site 214/215 on human PARP, demonstrating the staining of apoptotic cells by FITC-PARP CSSA is specific (lower right).

Product Laser Ex/Em Cat. No.  
Anti-PARP FITC Apoptosis Kit488495/529AHM2011product detail