synchronization and "Subnuclear Fractionation" of fibroblast - (Feb/14/2006 )
I have a couple questions about fibroblast cell:
1 how can I synchronize a fibroblast cell?
2 Are there possibilities to arrest such cultures in G1-, S-, G2-, or M-phase?
3 Did anyone get DNA associated proteins from fibroblast cell by Subnuclear Fractionation?how can I do it?
Thank you all guys.
My email: firstname.lastname@example.org
who will help me, thank you very much.
I know with mammalian cell lines that are in suspension can be "synchronized" by elutriation which separates the cells by centrifugation. Here's one paper that uses this method: http://www.pnas.org/cgi/reprint/93/12/5705
There are many drugs out there that can arrest cells in different phases. You might want to look up papers by Zbigniew Darzynkiew who is leader in flow cytometry. I found this link to some papers he's published on google.com: http://www.darzynkiewicz.com/zbigniew/mostcitedpapers.htm
I've read that the serum starvation is used for synchronization of cells, but I dont know how long does it take..I have HepG2 cells and in various articles people use starvation for different time...somebody during night, somebody for 2 or more hours..it's said their doubling time is 29 hours so I dont understand it.
By flow cytometry is possible to find out in which phase the cells are but I have no experience with it
Thank you two guys for your help. I will follow your instructions. Hope it works for me.
take on consideration that there are two very different things that until recenty were mistaken as one:
one thing is to synchronize a population of cells in culture
and another thing is arresting the cell cycle. In this case you will have cells in all stages of the cycle.
As obvious as it may sound to point out the difference, there were several papers in which serum starvation was used to synchronize cell populations, whose results were completely wrong.
if you apply a serum starvation protocol, you will be inducing the cells to enter in Gzero from the point of the cycle each one was, due to lack of nutrients, but if (for example) you treat them with colchicine, they will all stop cycling at the same point.
hope this helps...