Hi

You could start by staining for cyclin A2, that would roughly tell you if this localisation is associated with cycling or resting(+G1) cells. You could also try BrdU or EdU incorporation (followed by staining with resp. an antibody or a fluorescent azide - maybe to avoid destruction of the cilia by the acid required for BrdU treatment, you should choose EdU) to check if this occurs in S phase. Another classic thing to do is to stain for phospho-Histone 3 (although I doubt the cilia even exist in mitosis). Simply looking at DNA condensation by phase contrast would already indicate if the stained cells are in mitosis or not.

Good luck,

You might want to try this expression system from Life Tech... Premo™ FUCCI Cell Cycle Sensor (BacMam 2.0)Catalog number: P36237

Thanks for the answers, and Dr Ottman, thank you for the reference! I will try it, looks promissing.

Thanks for the answer.

I used the FACS and PI. 

The prmo Fucci is nice but I have a very low efficiency of transduction: can't get more than 5 % of cells expresing the system...

Does any of you experience in ciliation? The problem is now that I have seen the variation on the cell cycle of IMCD3, I need to make them ciliate in a more synchrouns way. Using Thymidine and/or Ro3306 is not helping, even if I combine with starvation media. Cells are in G1/S for ex with Thymidine block but still do not ciliate all at the same time and more important do not form cilia with the same final length... I have too much variation in cilai length at the control population. How can I improve this?

If any of you has experience on this, I appreciate any comment/ suggestion

Thanks in advance.

Hi Cecillia,

I'm running into the similar issue to this and I'm wondering if you ever landed on a successful method. I'm also working with IMCD3 cells and a protein that is a transient resident of cilia. In my hands IMCD3 are totally immune to any of the usual cell cycle synchronism methods. They proliferate rapidly and randomly in serum free media.