Measuring Primary Cilia Dynamics

mIMCD and MEF cells isolated from hArl13b-mCherry-GECO1.2^tg mice were seeded in an IBIDI µ-Slide VI 0.4 flow chamber coated with laminin (see IBIDI Application Note 11; for this chamber, apical membrane shear stress is: τ = η131.6 Φ: where η is dynamical viscosity (0.01 dyn s cm⁻²) and Φ is flow rate in ml/min. A syringe pump (Harvard Apparatus) delivered steady flow via 10 ml syringes containing L-15 medium. Z-stacks of primary cilia were recorded on an inverted Olympus FV1000 (60x, 1.2 N.A. water immersion objective). The bend angle was measured between ciliary base and tip^{18 (link)}. Fluid velocities were measured by imaging the flow of the solution supplemented with 300 nm green fluorescent beads (Sicastar greenF, Micromod) at the focal plane corresponding to ciliary tips at rest. Images were acquired as line scans (2 ms/line) or in continuous scanning mode (64 or 128 ms/frame) and particles tracked using an ImageJ plugin.