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Tools for Fluorescence-based Apoptosis and Caspase Detection

Apoptosis is an evolutionarily conserved form of cell suicide responsible for various biological processes including the normal turnover of cells and the proper functioning of the immune system. This process of shutting down old, diseased, or otherwise disfunctional cells is mediated by a cascade of proteolytic enzymes known as cysteine proteases, or caspases. Pro-apoptotic signals activate the enzymatic cascade resulting in the cleavage of protein substrates, leading to the disassembly of the cell (1-4). Caspases have been identified in organisms ranging from C. elegans to humans. Members of the mammalian caspase family of cysteinyl aspartate-specific proteases play distinct roles in apoptosis and inflammation.

Detection of activated caspase enzymes has much utility in basic and applied life science research, including the study of and the development of therapies against many cellular pathologies such as cancer and neurodegenerative disease. Among the research tools available for the qualitative and quantitative analysis of apoptosis-related proteins, fluorescence-based assays are useful for their flexible methods of analysis and multiplexing capabilities.

FLICA: Caspase Detection Assay Utilizing Fluorescent-Labeled Inhibitors
This fluorescence-based caspase detection assay utilizes a fluorescent inhibitor reagent that forms a covalent bond with active, intracellular caspase enzymes, preventing them from further catalysis and labeling them for fluorescence detection. The use of a blue nuclear dye, Hoechst 33342, and a red vital dye, Propidium Iodide, is optional to acquire additional information about nuclear morphology and cell membrane permeability (necrosis) in the assay.

Multiplexing with FLICA™: Propofol-treated hippocampal neurons exhibit cell death via caspase-3/7 activity, as detected by the FAM-FLICA Caspase 3/7 Assay Kit. Caspase-3/7-positive cells fluoresce green with the caspase probe FAM-DEVD-FMK while nuclear morphology is visualized with blue Hoechst 33342 DNA dye. Dying membrane-permeant cells fluoresce red with the vital dye Propidium Iodide. Data courtesy of Dr. Sibel Kahraman, U-MD School of Medicine.

 

ImmunoChemistry Technologies (ICT) developed this in vitro assay in the late 1990s, expanding upon the line with additional caspase targets and fluorophore labels. The assays for poly-caspases and for effector caspases-3-and-7 are sought after for their utility as overall indicators of apoptosis. An increasing number of citations for ICT’s protease detection products appear each month in peer-reviewed publications as research continues to discover new chemotherapeutic methods to accelerate cancer cell death or slow the progression of neurodegenerative processes.

Assess apoptosis and necrosis in cultured cells with FLICA™:

  • Sensitive and early indicator of the initiation of the apoptotic process
  • Distinguish apoptosis from necrosis
  • Flexible analysis methods
  • Green and red probes available
  • Detect poly caspases or individual caspase enzymes

Caspase Target; Use Fluorescence Kit
Poly Caspases; apoptosis FAM (green)
SR (red)
FAM-FLICA™ Poly Caspases Assay Kit
SR-FLICA™ Poly Caspases Assay Kit
Caspase 1 (ICE);
pyroptosis, inflammation
FAM (green) FAM-FLICA™ Caspase 1 Assay Kit
Effector Caspases 3/7;
apoptosis
FAM (green)
SR (red)
FAM-FLICA™ Caspases 3/7 Assay Kit
SR-FLICA™ Caspases 3/7 Assay Kit
Initiator Caspase 8;
extrinsic apoptosis pathway
FAM (green) FAM-FLICA™ Caspase 8 Assay Kit
Initiator Caspase 9;
intrinsic apoptosis pathway
FAM (green)
SR (red)
FAM-FLICA™ Caspase 9 Assay Kit
SR-FLICA™ Caspase 9 Assay Kit

See the full kit listing, data, and citations at www.immunochemistry.com/products/detection-reagents/flica.html.

References:
1. Slee, E. A., C. Adrain, and S. J. Maritin. (1999) Serial Killers: ordering caspase activation events in apoptosis. Cell Death Differ. 6:1067-1074.
2. Earnshaw, W.C., Martins, L.M., and Kaufmann, S.H. (1999) Mammalian caspases: structure, activation, substrates, and functions during apoptosis. Ann. Rev. Biochem. 68:383-424.
3. Hengartner, M.O. (2000) The biochemistry of apoptosis. Nature 407:770-816.
4. Degterev, A., Boyce, M., and Yuan, J. (2003) A decade of caspases. Oncogene 22:8543-8567.

 

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