title: Analysis of the spindle-assembly checkpoint in HeLa cells.
abstract: The spindle-assembly checkpoint involves signaling at kinetochores, which leads to the arrest of mitotic progression in the absence of microtubule attachment or spindle tension. Here, we detail procedures for the analysis of the spindle-assembly checkpoint in adherent mammalian cells. These techniques focus on pharmacological approaches and immunofluorescence microscopy to verify the state of spindle assembly, kinetochore attachment of microtubules and spindle tension, chromosome positioning, and kinetochore signaling by the Mad2 or Bub1 checkpoint proteins. We also describe a bi-parameter flow cytometric assay, using either MPM-2 or anti-phospho-(Ser10)-histone H3 antibodies, for quantitating mitotic cells.
authors.affiliation: Institut de Biologie Structurale, J-P Ebel (CEA-CNRS-UJF), Grenoble, France.
authors.forename: Paul R
authors.initials: PR
authors.lastname: Andreassen
authors.forename: Dimitrios A
authors.initials: DA
authors.lastname: Skoufias
authors.forename: Robert L
authors.initials: RL
authors.lastname: Margolis
mesh_terms: D002352:Carrier Proteins / Q000378:metabolism; D018797:Cell Cycle Proteins; D005434:Flow Cytometry / Q000379:methods; D006367:HeLa Cells; D006657:Histones / Q000276:immunology / Q000378:metabolism; D006801:Humans; D018386:Kinetochores / Q000737:chemistry* / Q000378:metabolism; D008856:Microscopy, Fluorescence / Q000379:methods; D008870:Microtubules / Q000378:metabolism; D008938:Mitosis / Q000502:physiology*; D009687:Nuclear Proteins; D011494:Protein Kinases / Q000378:metabolism / Q000502:physiology; D017346:Protein Serine-Threonine Kinases; D008941:Spindle Apparatus / Q000378:metabolism*; D014747:Vinblastine / Q000494:pharmacology
chemical_list: D002352:Carrier Proteins; D018797:Cell Cycle Proteins; D006657:Histones; D009687:Nuclear Proteins; D014747:Vinblastine; D011494:Protein Kinases; C584277:BUB1 protein, human; C090464:Bub1 spindle checkpoint protein; D017346:Protein Serine-Threonine Kinases