title: Monitoring the progression of cell death and the disassembly of dying cells by flow cytometry.
abstract: The use of annexin A5 (A5) and either propidium iodide or 7-aminoactinomycin D (PI/7-AAD) stains to measure cell death by flow cytometry has been considered the gold standard by most investigators. However, this widely used method often makes the assumption that there are only three types of particles in a sample: viable, apoptotic and necrotic cells. To study the progression of cell death in greater detail, in particular how apoptotic cells undergo fragmentation to generate membrane-bound vesicles known as apoptotic bodies, we established a flow cytometry-based protocol to accurately and rapidly measure the cell death process. This protocol uses a combination of A5 and TO-PRO-3 (a commercially available nucleic acid-binding dye that stains early apoptotic and necrotic cells differentially), and a logical seven-stage analytical approach to distinguish six types of particles in a sample, including apoptotic bodies and cells at three different stages of cell death. The protocol requires 1-5 h for sample preparation (including induction of cell death), 20 min for staining and 5 min for data analysis.
authors.affiliation: Department of Biochemistry and Genetics, La Trobe Institute for Molecular Science, La Trobe University, Melbourne, Victoria, Australia.
authors.forename: Lanzhou
authors.initials: L
authors.lastname: Jiang
authors.affiliation: Department of Biochemistry and Genetics, La Trobe Institute for Molecular Science, La Trobe University, Melbourne, Victoria, Australia.
authors.forename: Rochelle
authors.initials: R
authors.lastname: Tixeira
authors.affiliation: Department of Biochemistry and Genetics, La Trobe Institute for Molecular Science, La Trobe University, Melbourne, Victoria, Australia.
authors.forename: Sarah
authors.initials: S
authors.lastname: Caruso
authors.affiliation: Department of Biochemistry and Genetics, La Trobe Institute for Molecular Science, La Trobe University, Melbourne, Victoria, Australia.
authors.forename: Georgia K
authors.initials: GK
authors.lastname: Atkin-Smith
authors.affiliation: Department of Biochemistry and Genetics, La Trobe Institute for Molecular Science, La Trobe University, Melbourne, Victoria, Australia.
authors.forename: Amy A
authors.initials: AA
authors.lastname: Baxter
authors.affiliation: Department of Biochemistry and Genetics, La Trobe Institute for Molecular Science, La Trobe University, Melbourne, Victoria, Australia.
authors.forename: Stephanie
authors.initials: S
authors.lastname: Paone
authors.affiliation: Department of Biochemistry and Genetics, La Trobe Institute for Molecular Science, La Trobe University, Melbourne, Victoria, Australia.
authors.forename: Mark D
authors.initials: MD
authors.lastname: Hulett
authors.affiliation: Department of Biochemistry and Genetics, La Trobe Institute for Molecular Science, La Trobe University, Melbourne, Victoria, Australia.
authors.forename: Ivan K H
authors.initials: IK
authors.lastname: Poon
mesh_terms: D000818:Animals; D017304:Annexin A5 / Q000032:analysis*; D016923:Cell Death; D002460:Cell Line; D005434:Flow Cytometry / Q000379:methods*; D006801:Humans; D051379:Mice; D009696:Nucleic Acids / Q000032:analysis*; D013194:Staining and Labeling / Q000379:methods; D013997:Time Factors
chemical_list: D017304:Annexin A5; D009696:Nucleic Acids