Technical Reference #1858

1858.

Removal of Spindly from microtubuleattached kinetochores controls spindle checkpoint silencing in human cells Reto Gassmann; Andrew J. Holland; Dileep Varma; Xiaohu Wan; Filiz Cxivril; Don W. Cleveland; Karen Oegema; Edward D. Salmon; and Arshad Desai, University of California at San Diego - Ludwig Institute for Cancer Research, Genes & Development, 24(1858), (2010)
Link To Paper

Keywords:
Centromere; aneuploidy; mitosis; kinetochore; microtubule; spindle; chromosome

Materials & Methods:
For live-cell imaging experiments cells were seeded in a 35-mm glass-bottom dish coated with poly-D-lysine (MatTek). Cells were transfected using Oligofectamine and reduced-serum Opti-MEM (Invitrogen) according to the manufacturer’s instructions. A predesigned (Thermo Scientific) siRNA for Spindly (GA AAGGGUCUCAAACUGAA) or a nontargeting control siRNA (UGGUUUACAUGUCGACUAA) was used at a final concentration of 100 nM. After incubation for 5–6 h 1 vol of medium and fetal bovine serum (10% final) was added. After 24 h the transfection mixture was replaced with fresh medium. For immunofluorescence of HeLa Flp-In T-Rex cells transgene expression was induced with tetracycline 24 h post-transfection and cells were fixed 20–24 h later. For live-cell imaging of HeLa Flp-In T-Rex cells (and for the immunoblot shown in Fig. 1E) transgene expression was induced 22 h after transfection and the filming session was initiated 8 h later.

Microscopic Technique
Immunofluorescence

Cell Type(s)
HeLa and DLD-1 Flp-In T-Rex cell