Computational protocol: Effect of Cell Shape and Dimensionality on Spindle Orientation and Mitotic Timing

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Protocol publication

[…] HeLa-B (GFP-H2B/RFP-tubulin) cells were cultured in square or circular microwells for 18 hours before fixing by the addition of paraformaldehdye (4% (w/v) in PBS, Sigma-Aldrich, Switzerland) for 15 min and counter-stained by the addition of Vybrant DiD cell labeling solution (5 µM, 30 minutes, Molecular Probes). Images were subsequently captured on a Zeiss LSM510 confocal laser scanning microscope (Carl Zeiss AG, Germany) equipped with a 63× objective lens (1.4NA oil DIC Plan-Apochromat). Image stacks were captured using Zen software (Carl Zeiss AG) and mounted in figures using Imaris and Adobe Photoshop.HeLa-B (YFP-paxillin) cells were used to visualize the actin cytoskeleton after culture in the different shaped microwells and 2D square patterns. Consequently, HeLa-B (YFP-paxillin) cells were cultured on the different cell culture platforms for 18 hours before fixing for 3 min in warm 3% paraformaldehdye with Triton X-100 (0.5% (v/v), Fluka-Chemie AG) followed by 3% paraformaldehdye alone for an additional 40 min. Finally, HeLa-B (YFP-paxillin) cells were stained with phalloidin-TRITC (1∶200 dilution, Molecular Probes) and imaged using a Zeiss Live Cell Station microscope (Carl Zeiss AG) equipped with a camera (Hamamatsu) and 63× objective lens (1.4NA Oil DICIII Plan Apochromat). Image stacks were captured using Metamorph (Molecular Devices, Sunnyvale, CA) and deconvolved with Huygens Remote Manager and mounted in figures using Imaris and Adobe Photoshop. […]

Pipeline specifications

Software tools Imaris, MetaMorph
Application Laser scanning microscopy