transient overexpression and mitotic spreads

[pasquale pensieri]

Hello everyone,

I would glad if someone can give me an hint about my trouble...

I would like to transfect my cell line with a plasmid GFP-Protein of interest to overexpress it. Transfection should be transient and so, after a few generation I should lose my overexpressed protein in several cells.

In any case I should select GFP population after transfection with FACS and sorting, but on these cells I would like to do mitotic spreads... So I have to put the positive cells in plate for colcemid... Could you give me an hint? If I select a population GFP positive, how can I do later to get a good number of cells for a rapid colcemid treatment in time to mantain a good number of GFP cells so as not to miss plasmid and protein, but so as I could do a good number of spreads?   

I hope I was clear...

Thank you anyone...

Pasquale

[Nathan McCorkle]

It sounds like your actual question is this:

"""
If I select a population GFP positive, how can I do later to get a good number of cells for a rapid colcemid treatment in time to mantain a good number of GFP cells so as not to miss plasmid and protein, but so as I could do a good number of spreads?

"""

What organism?

FACS is very fast, "later" should be a fraction of the cell division (doubling) time length. Depending on the FACS you have available, you may be able to "bin" your positive cells with respect to brightness... cells that are dim have either only just started to express, or are already losing the plasmid... so I would guess you want the cells around the peak of the histogram (of brightness).

If you still have issues, I might look into the possibility of setting up a feedback loop in the FACS... where the waste/negative-cells (cells which are not bright) are fed back into the input (in case the cells have not begun to express yet, you can keep them looping in the FACS until they just start to express). The challenge here, I think, will be re-adjusting the growth media/buffer, since the sheath fluid likely will affect growth rate. You might need to implement some sort of in-line centrifugation or dialysis, so you can keep the cells happily growing.