Fig. 1.

Experimental procedure for measuring CBF and cilia beating direction in ependymal cultures treated with fluid flow. Continuous fluid flow is applied for 3 days on cells cultured in Transwell chips. The chips are then removed from the flow and cilia motility is imaged with the microscope. From high-speed movies in bright field (BF) illumination, we identified ciliary beating frequency and cell position, while from the motion of tracer particles we inferred the ciliary beating direction. (A) Schematic of an ependymal culture in a Transwell chip. (B) Illustration of the setup: shear stress is applied on cells by cycling medium in a closed loop using a peristaltic pump and a medium reservoir. (C,D) Normalised standard deviation maps of the pixel intensity over time, from a BF movie (C) and fluorescence (FL) images after the addition of tracer particles (same field of view) (D). (E) The information from BF and FL movies merged. The colour map indicates the ciliary beating frequencies (CBF) and the red arrows the measured flow field above the identified cells. The length of the arrows is proportional to the flow velocity. The analysis procedure is also explained in Movie 1.