title: Measuring caspase activity by Förster resonance energy transfer.
abstract: Förster resonance energy transfer (FRET) occurs across very short distances (in the nanometer range) between donor and acceptor fluorophores that overlap in their emission and absorption spectra. FRET-compatible green fluorescent protein (GFP) variants that are fused to short peptide linkers containing caspase cleavage sites can be used to measure caspase activity. In the intact probes, the donor and acceptor fluorophores are in close proximity, and FRET is highly efficient. On caspase activation, proteolysis of the linker occurs, and the donor is separated from the acceptor. This results in a disruption of resonance energy transfer and an increase in donor fluorescence quantum yield; this event is typically referred to as sensitized emission or donor unquenching. A number of highly sensitive FRET probes based on the cyan fluorescent protein-yellow fluorescent protein (CFP-YFP) pair, or improved variants thereof, have been developed to detect intracellular caspase activities. In this protocol we describe how to use FRET-based caspase substrates and time-lapse imaging to measure caspase activity in cells undergoing apoptosis.
authors.affiliation: Centre for Systems Medicine, Royal College of Surgeons in Ireland, Dublin 2, Ireland Department of Physiology and Medical Physics, Royal College of Surgeons in Ireland, Dublin 2, Ireland.
authors.forename: Markus
authors.initials: M
authors.lastname: Rehm
authors.affiliation: Center for Cell and Gene Therapy, Baylor College of Medicine, Houston, Texas 77030 Department of Pediatrics-Hematology, Baylor College of Medicine, Houston, Texas 77030.
authors.forename: Melissa J
authors.initials: MJ
authors.lastname: Parsons
authors.affiliation: Center for Cell and Gene Therapy, Baylor College of Medicine, Houston, Texas 77030 Department of Pediatrics-Hematology, Baylor College of Medicine, Houston, Texas 77030.
authors.forename: Lisa
authors.initials: L
authors.lastname: Bouchier-Hayes
mesh_terms: D000818:Animals; D017209:Apoptosis / Q000502:physiology; D020169:Caspases / Q000378:metabolism*; D002478:Cells, Cultured; D031541:Fluorescence Resonance Energy Transfer / Q000379:methods*; D006801:Humans; D007091:Image Processing, Computer-Assisted; D008164:Luminescent Proteins / Q000235:genetics / Q000378:metabolism; D018613:Microscopy, Confocal; D008856:Microscopy, Fluorescence; D013997:Time Factors
chemical_list: D008164:Luminescent Proteins; D020169:Caspases