From owner-cellbiol@net.bio.net Tue Jan 21 22:00:00 1997 Path: biosci!ZOOL.UMD.EDU!goode From: goode@ZOOL.UMD.EDU ("Dennis Goode") Newsgroups: bionet.cellbiol Subject: Re: cell cycle synchronization Date: 22 Jan 1997 08:51:13 -0800 Organization: University of Maryland Zoology Lines: 56 Sender: daemon@net.bio.net Distribution: world Message-ID: NNTP-Posting-Host: net.bio.net Stephan Niland (100766.3150@compuserve.com) <100766.3150@compuserve.com> wrote on the subject: cell cycle synchronization >Hi, out there, >I ve got three questions on cell culture synchronization: >1)how can I best synchronize a fibroblast cell culture? >Is mitotic shake-off really the best, or are there any >protocols using certain drugs? If yes, please mail me >the respective reference or protocol! Stephan, This depends on what you want to do. Mitotic shake off is good for studying mitotic and early G1 events in a small population of cells, because it can give you over 90% of the cells in mitosis. But if you need to do biochemistry on a large number of cells, then you need a method that synchronizes the entire population. No method gives you completrely synchronous cells. Nocodozole blocked and selected cells move thru mitosis quite synchronously after removel of the Nocodazole (Zieve et al., Exp. Cell Res. 126:397.) >2) How long will the culture probably be synchronous? Only for a short period of time. Most cells lose synchrony in G1 because it is the most variable phase, even in constant cell culture conditions. >3) Are there possibilities to arrest such cultures in G1-, > S-, G2-, or M-phase? if yes, how can I do it? Cell cycle researchers most commonly synchronize cells at the G1-S boundary with reversible inhibition of DNA synthesis. One way is the double excess-thymidine block, that has the advantage of being non-toxic and very reversible. These cells stay fairly well synchronized until the next G1, so are good for S, G2 and even M phase events. Also one can select cells in partyicular phases with FACS (automated cell sorter). See Mitchison, The Biology of the Cell Cycle Zeuthen, Synchrony in Cell Division and Growth Ahearne, An intoduction to Cell Population Kinetics. >I would be glad, if you could help me, and I am looking >forward to receiving your mail! >Stephan (e-mail: 100766.3150@compuserve.com) -Dennis Dr. M. Dennis Goode Phone (301) 405-6917 Department of Zoology Fax (301) 314-9358 University of Maryland e-mail goode@zool.umd.edu College Park MD 20742 ************************************************************* "If the Lord Almighty had consulted me before embarking upon the creation, I should have recommended something simpler." -Alphonso X of Castile, 15th Century .