After keeping the cells at 100% confluency for 1 d (a condition that arrests the cells at the end of G1; Coupin et al., 1999 (link); Kozik et al., 2010 (link)), they were trypsinized and seeded onto 5-mm-diameter coverslips (Warner Instruments) that were cleaned by sonication in 95% ethanol at ∼30–50% confluency; this procedure helped to synchronize cell division and increase the proportion of cells entering S phase. The cells in prometaphase and metaphase were identified by their spherical appearance 11–16 h after plating. The cells were then imaged in Leibovitz’s L-15 medium without phenol red (21083-027; Life Technologies) supplemented with 5% FBS and 20 mM HEPES, pH 7.4.
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Cell Synchronization for Live Imaging
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Other organizations : Harvard University, Boston Children's Hospital, Janelia Research Campus, Howard Hughes Medical Institute, Center for Systems Biology, Philadelphia University, The University of Texas Southwestern Medical Center
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Variable analysis
independent variables
- Cell confluency (100%)
- Cell seeding density (∼30–50% confluency)
- Proportion of cells entering S phase
- Cells kept at 100% confluency for 1 day
- Cells were trypsinized and seeded onto 5-mm-diameter coverslips
- Cells were imaged in Leibovitz's L-15 medium without phenol red, supplemented with 5% FBS and 20 mM HEPES, pH 7.4
- No positive or negative controls were explicitly mentioned.
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