I'm using the fucci bacmam 2.0 cell cycle reporter kit to describe cell cycle dynamics in a stem cell line, which we think last for about 30h. The problem is that transfected cells die much earlier than that, and those that don't, seem to have no cycling (allways express the same reporter, continuosly). I have tested it also in HeLa cells, and it seems to work better, but I still cannot find any cell that completes a whole cycle (and I've analyzed some 300+ cells). Since HeLa are suppose to complete a division every 24h (+/-) hours, I'm wondering if the system works well at all, for what I intend to do...
Is anyone using it for long live imaging experiments? If so, do you find any delay in cell cycling? Do your cells still divide after transfection? And do you also have strong cytoplasmic expression of the gemining-GFP reporter?
Thank you for your help
Hi, I am starting to use Fucci plasmids but not for live imaging. I've sublocned mAG-hGeminin and mKO2-hCdt1 into lentiviral plasmid, tested them by transient transfection in HEK293t and I see both of them nuclearly localized. Now I am transducing mouse embryonic stem cells to generate stable cell lines. I am also selecting cells with two different antibiotics (Puro and Hygro) and it seems to work. I noticed a differentiation phenotype after mOK2-hCdt1 transduction and I slight delay in proliferation, but it's just speculation, I didn't measure it yet., Anyway cells seems to have recovered after splitting them. There are publications in which they've succesfully achieved live cell imaging of dividing mESCs with the FUCCI reporter, it should work pretty fine. My suggestion is to try to generate stable cell lines, this could solve your issue I guess.
Good luck
I'am pretty sure that the FUCCI approach works well. You observe cells with constant expression of the reporter because the expression levels exceede the capacity of the E3 ligases. The high expression levels resulting from transient expression are clearly the probem, so it is highly recomended to generate stable cell lines. Luckily are now plenty of FUCCI-expressing cell lines (see table2 of our review) available to test your live imaging protocol.
Article FUCCI sensors: powerful new tools for analysis of cell proli...
Hi Goncalo,
I will also confirm that it does work, but you do have to use stable cell lines. We work with cancer stem cells, in both normal and hypoxic conditions. The system works fine for us both as static measurement of cell cycle status and in live cell imaging.
Kind regards,
Steve
A short update, just finished constructing stable cell lines, it works great ;)
Hello everyone
Thank you for the help. Yes, creating a stable cell line has been discussed previously aswell. The problem with that is that I'm using a cell line with very few usefull passages (around 10), and I am also doing infections to create stable expression of other constructs. The idea of using a trasient Fucci expression was really to not waste anymore passages in infection/selection.
The transient Fucci I'm using comes in a ready-to-use mix from Thermo Scientific. It was suggested to us by a colaborator that apparently used it and liked it. But, atleast for long periods of live imaging, it appears to work rather badly, even in HeLa. I guess stable line is the way to go, for better or worse.
Thank you again for the help!
I used Jurkat T cells and LIM1215 cells. However, to my disappointment, instead of staining the nuclear area, it seems that the stain is stained on organelle around the nucleus. Which is very confusing. The labelling volume I used is 1 mL. 20μL component A + 20μL component B, 16 hrs incubation time.
Hi All
I would like to have FUCCI sensor plasmids to package into lenti virus. If anyone can give me small amount of plasmids. I highly appreciate it.
Thank you in advance.
Pooja
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