Hi Ronald Given the choice, I would go for the Hoechst to avoid the problems of fixation etc with 7AAD (and the fact that Hoechst will give the better histogram). The protocol for Hoechst staining for DNA is quite straightforward although the concentration used and the time of staining will depend on cell type and what you want to do with them afterwards. I find that 10ug/ml Hoechst 33342 for 20-3omins at 37C is a good place to start. The optimal conditions can then be determined empirically for your cell type. The dye is a bit cytotoxic so if you want to re-grow, you should be careful about the amount of dye. Its a balance between getting a good DNA profile and keeping the cells alive. Of course if you are doing quantitative DNA staining you will want to use pulse processing to remove G1 doublets. I am willing to be corrected but doesnt the Vantage only allow 7 live parameters to be acquired? So using two scatter parameters, 4 colours and Hoechst wouldnt allow the PP boards to be included. Maybe you could sacrifice SSC and use A v H of the Hoechst signal to gate for single cells. Presumabaly the Vantage SE will allow more parameters to be acquired! Hope this helps!! Derek On Mon, 17 May 1999, Ronald Rabin wrote: > I am planning to do 4 color analysis + cell cycle on a dual laser Vantage > that can use either a dye laser or a UV laser as the second laser. I can > use either FITC, PE, PECy5, Red613 and Hoechst, or I can use FITC, PE, 7ADD, > R613, and APC. Because my PE conjugates are critical to the experiment, I > cannot use the Pyronin Y. It seems the Hoechst would be easier, cell > permeable etc, though I have no experience with it. **************************************************************************** * 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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