The FRAP assay was performed with adaptations from Huis in ’t Veld et al. (2014) (link) and Chan et al. (2012) (link) and are described in the Supplemental Material . Cells were synchronized by 100 ng/mL nocodazole for 7.5 h and then released into medium containing 0.5 mM of auxin. Five hours or 6 h after release, 40 µL of cells was transferred into 15 µ-slide angiogenesis ibiTreat chamber slides (ibidi 81506), and remaining cells were fixed by 70% EtOH for cell cycle analysis by propidium iodide staining. During imaging, cells were cultured in normal medium at 39.5°C and 5% CO2.
Protocol detail
Synchronized Cell Imaging of FRAP
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Adaptations
Angiogenesis
Assay
Auxin
Cell cycle
Cells
Cultured medium
Etoh
Nocodazole
Propidium iodide
Corresponding Organization :
Other organizations : FIRC Institute of Molecular Oncology, Tokyo Metropolitan University, Istituto di Genetica Molecolare
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Variable analysis
independent variables
- Nocodazole treatment (100 ng/mL) for 7.5 h
- Auxin treatment (0.5 mM) after release from nocodazole
- Cell cycle progression
- Temperature (39.5°C)
- CO2 concentration (5%)
- Positive control: Not specified
- Negative control: Not specified
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