title: Parallel single-cell analysis of active caspase-3/7 in apoptotic and non-apoptotic cells.
abstract: Analysing the chemical content of individual cells has already been proven to reveal unique information on various biological processes. Single-cell analysis provides more accurate and reliable results for biology and medicine than analyses of extracts from cell populations, where a natural heterogeneity is averaged. To meet the requirements in the research of important biologically active molecules, such as caspases, we have developed a miniaturized device for simultaneous analyses of individual cells. A stainless steel body with a carousel holder enables high-sensitivity parallel detections in eight microvials. The holder is mounted in front of a photomultiplier tube with cooled photocathode working in photon counting mode. The detection of active caspase-3/7, central effector caspases in apoptosis, in single cells is based on the bioluminescence chemistry commercially available as Caspase-Glo®3/7 reagent developed by Promega. Individual cells were captured from a culture medium under microscope and transferred by micromanipulator into detection microvial filled with the reagent. As a result of testing, the limits of detection and quantification were determined to be 0.27/0.86 of active caspase-3/7 content in an average apoptotic cell and 0.46/2.92 for non-apoptotic cells. Application potential of this technology in laboratory diagnostics and related medical research is discussed. Graphical abstract Miniaturized device for simultaneous analyses of individual cells.
authors.affiliation: Institute of Analytical Chemistry, v.v.i., Czech Academy of Sciences, Veveří 97, 60200, Brno, Czech Republic.|Faculty of Science, Masaryk University, Kotlářská 267/2, 61137, Brno, Czech Republic.
authors.forename: Vojtěch
authors.initials: V
authors.lastname: Ledvina
authors.affiliation: Faculty of Science, Masaryk University, Kotlářská 267/2, 61137, Brno, Czech Republic.|Institute of Animal Physiology and Genetics, v.v.i., Czech Academy of Sciences, Veveří 97, 60200, Brno, Czech Republic.
authors.forename: Eva
authors.initials: E
authors.lastname: Janečková
authors.affiliation: Institute of Animal Physiology and Genetics, v.v.i., Czech Academy of Sciences, Veveří 97, 60200, Brno, Czech Republic.|Department of Physiology, University of Veterinary and Pharmaceutical Sciences, Palackého 1/3, 61242, Brno, Czech Republic.
authors.forename: Eva
authors.initials: E
authors.lastname: Matalová
authors.affiliation: Institute of Analytical Chemistry, v.v.i., Czech Academy of Sciences, Veveří 97, 60200, Brno, Czech Republic. kleparnik@iach.cz.
authors.forename: Karel
authors.initials: K
authors.lastname: Klepárník
mesh_terms: D000818:Animals; D017209:Apoptosis; D053148:Caspase 3 / Q000032:analysis / Q000378:metabolism*; D053179:Caspase 7 / Q000032:analysis / Q000378:metabolism*; D002478:Cells, Cultured; D057075:Enzyme Assays / Q000295:instrumentation* / Q000379:methods; D004867:Equipment Design; D008163:Luminescent Measurements / Q000295:instrumentation* / Q000379:methods; D051379:Mice; D059010:Single-Cell Analysis / Q000295:instrumentation* / Q000379:methods
chemical_list: D053148:Caspase 3; D053179:Caspase 7
keywords: Apoptosis; Bioluminescence; Caspase-3/7; Single-cell analysis