serum starvation - induce cilia by serum starvation in Rpe-1 cells (Jun/11/2007 )
Hi everyone,
I've been trying serum starvation (0.25% FBS) to induce cilia in hTert-RPE-1 cells but couldn't success. I got like only 5-10 cells with induced cilia per slide, which is about 90-95% confluent when I fixed the cells. The amount of FBS (0.25%) is based on literature. I grow the cells for few days to reach ~80% confluent and then change media with only 0.25% FBS and let it set for 48-72 hours. Since I could not successfully induce cilia, I would like to ask if anyone is familiar with this cell line and know the tips of cilia induction. I really appreciate it. Thanks a lot.
I'm not familiar with this cell line, but two things that come to mind are (1) double check your FBS concentration, or just re-make the media to be sure, and (2) you may want to try washing with warm PBS before adding your reduced-serum media.
Thanks Cassio,
But those are what I have done. I add FBS before I did this experiment and also wash the cells with PBS (but not warm, just room temp.) before adding reduced-serum media. Do you know if the cell density will make the difference?
Our RPE-1 cell has primari cilia in complete media,we do,nt need to let it in media wiht low serum, but if I let in media with 0,5% FBS and letting it for 24 hours there is a lot of primari cilia, and bigger than standar conditions.
There is no problem to induce primari cilia.
I detect primari cilia using 611B antibody, which antibody do you use?
I think we could exchange cells, you could get RPE-1 with cilia and i could get RPE-1 without it.
Sorry for my bad english.
There is no problem to induce primari cilia.
I detect primari cilia using 611B antibody, which antibody do you use?
I think we could exchange cells, you could get RPE-1 with cilia and i could get RPE-1 without it.
Sorry for my bad english.
Actually, I finally induced cilia, which I think it has to be 100% confluent to produce cilia. It is interesting that you don' t have to serum starve cells to get cilia. Aren't we supposed to have same cell line, hTert-RPE1? or it is because my passage number is high (e.g. passage 30 or more) when I did serum starvation? (PS. our cells is a gift from other lab though.)
The antibody I used is mouse anti-acetylated tubulin (Sigma).
Actually, I finally induced cilia, which I think it has to be 100% confluent to produce cilia. It is interesting that you don' t have to serum starve cells to get cilia. Aren't we supposed to have same cell line, hTert-RPE1? or it is because my passage number is high (e.g. passage 30 or more) when I did serum starvation? (PS. our cells is a gift from other lab though.)
The antibody I used is mouse anti-acetylated tubulin (Sigma).
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I think we have the same cell line but I don't use cells if the passage number is over 20, this is very important. Our RPE-1 are from other lab too, from Paris. In fact is very interesting your RPE-1 if in standars conditions they have not primari cilia.
20? I thought it is infinite cell line and could be used for at least 30 passages. Our stocks are already passage 10 though. But I have thawed a new batch of cells and will see if they have cilia without serum starvation. Thanks a lot for your information.
20? I thought it is infinite cell line and could be used for at least 30 passages. Our stocks are already passage 10 though. But I have thawed a new batch of cells and will see if they have cilia without serum starvation. Thanks a lot for your information.
Have you seen if your cells have cilia without serum starvation?