Hi Ken, The way that we do this is to use Hoechst 33342. However you are right about the toxicity effect. You generally need to play about with the concentration of the dye and the staining time to get the balance right between getting enough dye in to get a decent profile to enable you to sort and not killing the cells. I would hazard a guess that the concentration would be between 5-10ug/ml and the time no longer than 30 mins but it will vary with cell type. Using Hoechst to define a population to be sorted is fine but using it as a method of synchronising cycle phase is a little more problematical. Cells do tend to lag a little after sorting - some dont like it at all, of course - and the dye will take some time to be removed from the cells. We have had some success using centrifugal elutriation to select cell cycle phases so that is an alternative non-flow approach! Derek On Sun, 24 Jan 1999, KMCDONALD wrote: > Does anyone know a method of staining cells for DNA cell > cycle analysis with the intension of sorting the various phases and > culturing them. I can only think of using Hoechst staining (sterile) and > then sorting, however I've heard that Hoechst can be toxic. > Thanks for your responses in advance...Ken McDonald **************************************************************************** * Derek Davies Voice: (44) 0171 269 3394 * * FACS Laboratory, FAX: (44) 0171 269 3100 * * Imperial Cancer Research Fund, e_mail: derek.davies@icrf.icnet.uk * * London, UK * * * * Web Page: http://www.icnet.uk/axp/facs/davies/index.html * ****************************************************************************
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