Video for: The role of hydrodynamic forces in synchronisation and alignment of mammalian motile cilia

<p>Here you can find all the videos of the PhD thesis: &nbsp;</p> <p>The role of hydrodynamic forces in synchronisation and alignment of mammalian motile cilia.</p> <p>by Nicola Pellicciotta</p> <p>Video1:&nbsp;This example shows a movie of a single cell, being subjected to an external oscillatory flow. Images were acquired at 500\,fps using a 60X objective, then analysed using background subtraction and spatial median filter of 3x3 pixels. &nbsp;Chapter 3</p> <p>Video 2: &nbsp;we show the entrainment of a group of cells with very strong oscillatory external flow $v_\mathrm{EX}= 2$\,mm/s at $f_\mathrm{EX}=12$\,Hz. This s flow induced entrainment and also alignment of beating direction of some cells that were beating misaligned to the external flow. The original direction of beating was recovered after the flow stopped.&nbsp;Chapter 3</p> <p>Video3 and Video4: we depolymerised cell actin by adding 2uM Cytochalasin-D in the cell culture medium for 48hr. After imaging, cells (control and dug treated) were stained in 4\% PFA for 10 minutes, permeabilised with Triton x-100 0.1\% in PBS, and incubated for 1 hour with Nucblue R37605 (1 drop for mL of PBS) and with phalloidin for actin (Sir-Actin, 0.2 uM) following proprietary protocols. Z-stack were taken with confocal microscope (slices of distance of 0.15um each) and can be found at the following links: DMSO treated cells (control) Video 3, and Cytochalasin-D treated, &nbsp;Video 4.&nbsp;Chapter 3</p> <p>Video5:&nbsp;The maximal beating amplitude for a cilium within each cell was measured by inspecting the recordings from top view and marking two extreme points at the power and recovery stroke.&nbsp;Chapter 3</p> <p>Video6: Experimental procedure &nbsp;and the image analysis methods for Chapter 4.&nbsp;Continuous fluid flow is applied for three 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 Bright Field movies we identify ciliary beating frequency and cell position, while from tracer particles we measure ciliary beating direction.&nbsp;</p> <p>Video7: Video of&nbsp;Propelled particles&nbsp;in a culture at 29 DIV and treated with shear stress &tau;&nbsp;&asymp;0.8 dyne/cm2. Chapter 4</p> <p>&nbsp;</p>