title: Bioluminescence determination of active caspase-3 in single apoptotic cells.
abstract: Caspase-3 is an executive caspase, in the central position within apoptotic machinery. Apoptosis as a way of programmed cell death is a physiological process that plays an essential role in the development and homeostasis maintenance; moreover, its deregulations are linked to tumor progression or various autoimmune disorders. Therefore, an investigation of apoptosis pathways on the level of individual cells is not only of biological but also medical importance. In this work we report on the development of a high-sensitivity instrumentation and protocol for detection of active caspase-3 in individual mammalian apoptotic cells. The technology is based on the specific cleavage of modified luciferin by caspase-3, an immediate bioluminescence reaction of free luciferin with luciferase followed by emissions of photons and their detection by photomultiplier tube working in the photon counting regime. Three different instrumental arrangements are compared for the determination of caspase-3 in free cells or tissue samples. Thus, in our best miniaturized system the mean amount as low as about 6.5 fg corresponding to 122 000 molecules of caspase-3 can be detected in individual apoptotic mouse leg cells.
authors: Institute of Analytical Chemistry, v.v.i., Czech Academy of Science, Brno, Czech Republic.
authors: Marcela
authors: M
authors: Lišková
authors: Karel
authors: K
authors: Klepárník
authors: Eva
authors: E
authors: Matalová
authors: Jitka
authors: J
authors: Hegrová
authors: Jan
authors: J
authors: Přikryl
authors: Eva
authors: E
authors: Svandová
authors: František
authors: F
authors: Foret
mesh_terms: D000818:Animals; D017209:Apoptosis / Q000502:physiology*; D053148:Caspase 3 / Q000032:analysis* / Q000378:metabolism*; D002478:Cells, Cultured; D004797:Enzyme-Linked Immunosorbent Assay; D005260:Female; D006801:Humans; D008163:Luminescent Measurements / Q000295:instrumentation / Q000379:methods*; D051379:Mice; D011247:Pregnancy; D059010:Single-Cell Analysis / Q000295:instrumentation / Q000379:methods*
chemical_list: D053148:Caspase 3