I use the most commonly used cilia marker, acetylated alfa tubulin, to stain primary cilia. With the help of confocal microscope I take a z-scan image of cilia. I, however, looking for a software where I can automatically count cilia and measure their length. Any ideas?
There are reasons to believe that measurements in the z-axis are not as accurate as the x- or y- axes due to motor calibrations and overlapping sections with different pinhole dimensions. Since you're trying to measure primary cilia (one cilium per cell) it should not be cumbersome to measure a few cilia especially if you manage to image them in the x- or y- axes using ImageProPlus or calibrated software. I guess the trick would be to place them en-face withouth causing too much shrinking during fixation. - Just some thoughts.
Thanks, it is indeed imprecise measuring the z-axis. However, with polarised cells grown in a semi-permeable membrane transwells, the cilia appear in an upright position thus are seen as dots in an x-y plane, which requires taking a z-scan. Is there a way I can make them flat and measure them in an x-y plane? As for fixation, 30 min 4% paraformaldehyde doesn't cause shrinking (I hope) !!!
My experience is with human bronchial epithelial cells and they have a lot of cilia. En-face imaging certainly causes shrinking but in general en-face processing it's unreliable. Since you need to remove the Transwell membrane from the insert prior to immunostaining, I'd suggest cutting the membrane out PRIOR to fixation, cut a pizza slice for trypsinization and leave the rest of the pizza for formalin fixation. With the unfixed pizza slice, put it in a 1.5 mL tube and trypsinize for a few minutes, neutralize with serum, resuspend disgregated cells in 200 uL of media with serum, and centrifuge them unto a microscope slide using a Cytospin centrifuge (don't load more than 20,000 cells per cytospin well). Spin 5 min and then mark a circle around cytospun cells with hydrophobic pen and fix with formaldehyde as before. Try imaging clilia using the z-stack approach as well as the cilia that you find in the x or y planes.
There is a publication from Sue McGlashan's team in New Zealand which provides a method to address this question. Please see below:
Saggese T, Young AA, Huang C, Braeckmans K, McGlashan SR. Development of a
method for the measurement of primary cilia length in 3D. Cilia. 2012 Jul
3;1(1):11. doi: 10.1186/2046-2530-1-11
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