Computational protocol: Regulation of Cilium Length and Intraflagellar Transport by the RCK-Kinases ICK and MOK in Renal Epithelial Cells

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Protocol publication

[…] Clonal IMCD-3 cells stably expressing mCit-KIF3B, IFT43-YFP, GFP-BBS8, IFT20-GFP, KIF17-mCit, GFP-ICK or GFP-MOK were grown on 18 mm cover slips. Prior to analysis glass slides were inversed onto a 24 mm cover slip and placed in a live-cell imaging chamber. Time-lapse movies were acquired on a spinning-disc microscope (CSU-X1-A1; Yokogawa) equipped with 100×1.49 NA oil objective (Nikon) and an EMCCD camera (QuantEM 512SC; Roper Scientific), installed on an inverted research microscope (Eclipse Ti-E; Nikon), and controlled with MetaMorph 7.5 software (Molecular Devices). To determine the IFT particles' velocities, kymographs were generated in ImageJ with the Kymograph plugin, written by J. Rietdorf. […]

Pipeline specifications

Software tools MetaMorph, ImageJ
Applications SPIM, Microscopic phenotype analysis
Organisms Mus musculus, Homo sapiens
Chemicals Sirolimus