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 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 green FAM-FLICA Caspase 3 Assay. Caspase-3/7-positive cells fluoresce green with the caspase-3 & 7 inhibitor 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 caspase assay in the late 1990s, expanding upon the line with additional caspase targets and fluorophore labels. The assays to detect poly-caspase activity and 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 and apoptosis 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 caspase activity or individual active caspase enzymes

Caspase Target		Fluorescent Label		Caspase Detection Assay
Poly Caspases; apoptosis		FAM (green) SR (red) 660 (far-red)		FAM-FLICA™ Poly Caspase Assay Kit SR-FLICA™ Poly Caspase Assay Kit FLICA® 660 Poly Caspase Assay Kit
Caspase 1 (ICE); pyroptosis, inflammation		FAM (green) 660 (far-red)		FAM-FLICA™ Caspase 1 Assay Kit FLICA™ 660 Caspase 1 Assay Kit
Caspase 2; regulator		FAM (green)		FAM-FLICA™ Caspase 2 Assay Kit
Caspase 3/7; effectors, apoptosis (DEVD-ases)		FAM (green)		FAM-FLICA™ Caspase 3/7 Assay Kit
Caspase 6; effector, apoptosis		FAM (green)		FAM-FLICA™ Caspase 6 Assay Kit
Caspase 8; initiator, extrinsic apoptosis pathway		FAM (green)		FAM-FLICA™ Caspase 8 Assay Kit
Caspase 9; initiator, intrinsic apoptosis pathway		FAM (green) SR (red)		FAM-FLICA™ Caspase 9 Assay Kit SR-FLICA™ Caspase 9 Assay Kit
Caspase 10; initiator, extrinsic apoptosis pathway		FAM (green)		FAM-FLICA™ Caspase 10 Assay Kit
Caspase 13; effector, apoptosis		FAM (green)		FAM-FLICA™ Caspase 13 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.