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Life Technologies' Caspase Assays for Flow Cytometry

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

Live Cell Caspase Assays

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.

 

ConjugateCaspase
Detected
LaserEx/EmCat. No.  
Vybrant® FAM™ Poly caspases assay kit All 488 495/529 V35117 product detail
Vybrant® FAM™ caspase-3 & -7 assay kit 3,7 488 495/529 V35118 product detail
Vybrant® FAM™ caspase-8 assay kit 8 488 495/529 V35119 product detail

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).

ProductLaserEx/EmCat. No.  
Anti-PARP FITC Apoptosis Kit 488 495/529 AHM2011 product detail