8 replies to this topic

[cardosopedro]

Hey.

I want to measure the distribution of the cell cycle in my cells. But I have a big problem. My cells have more than one nuclei (usually 4-5) and the cell cycle is asynchronous. Therefore, within the same cell the different nuclei are in different phase of the cycle.

So, I can't use the traditional propidium iodide staining of DNA/flow cytometry to analyse this. Can anyone indicate me an alternative method for this?


Thanks.

[bob1]

Could you try synchronising the cells by serum starvation or a chemical means?


Out of curiosity:  how do you know the difference nuclei are in different stages of the cell cycle?

[cardosopedro]

Hey, thanks for the answer.

I think I could synchronize the cells by chemical means. But then how would I evaluate the effects of drugs in cell cycle distribution?

Regarding the other question, I simply looked in the literature and saw that these nuclei divide asynchronously.

[bob1]

I think the answer is to switch to mono-nucleate cells

You could make the assumption that each individual nucleus is a "cell" and work from there?


Thanks for the info re the nuclei, that's pretty interesting... I wonder how it works for cell division?

Edited by bob1, 13 May 2011 - 04:27 PM.

[cardosopedro]

Thanks again for the answer.

What do you mean by "switch to mononucleate cells"? Change the type of cells I am working with? That is not possible, because I am working with a fungus.

Yes, it is interesting how the cells can orchestrate asynchronous cell division.

[bob1]

Ah, fungus makes sense... I assumed that you were working with a mammalian cell line, like most of the questions on here, which occasionally present as multi-nucleate.

I have just realised that treating each nucleus as a cell may not work either - they may be too close together to measure the different nuclei, and you certainly wouldn't be able to cell sort, if you were aiming for that.

[cardosopedro]

mmmm... yes, I know, sometimes is hard to work with my fungus...

Do you have any other suggestion to evaluate cell cycle?

Thanks.

[bob1]

I guess you will have to look for cell cycle markers by immunological methods, but that will be very laborious unless you could use that for multichannel FACS.  You could try contacting a company like Dako who might be able to help more.

[cardosopedro]

Ok, thanks.