Analysis of the spindle-assembly checkpoint in HeLa cells.
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.
Andreassen
Paul R
PR
Institut de Biologie Structurale, J-P Ebel (CEA-CNRS-UJF), Grenoble, France.
Skoufias
Dimitrios A
DA
Margolis
Robert L
RL
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
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