Hi there Are you gating on 7AAD positive or negative cells in your analysis? I am using 7AAD as an exclusion dye together with surface staining (rather than cell cycle analysis), but hopefully the principles are the same. 1) I use single colour controls for fitc and pe, and individually remove excess FL1 and FL2 fluorescence from the FL3 channel. 2) I do not remove FL3 out of FL2. The reason is because I am using 7AAD as an exclusion dye, ie. in my analysis I gate on the 7AAD negative population. Since 7AAD negative cells have no fluorescence they will not contribute excess fluorescence into other channels (and therefore do not need to be compensated). I hope this helps. Lydene McArthur Haematology Surface Markers department Canterbury Health Laboratories Christchurch New Zealand >>> "Kotsianidis, Ioannis" <i.kotsianidis@imperial.ac.uk> 01/27/05 04:29a.m. >>> Dear Flowers, I am trying to perform 7-AAD staining for cell cycle with simultaneous surface staining. When I tried to compensate 7-AAD (linear, FL3) with PE (log, FL2), 1) I wasn't able to see any event on an FL2 vs FL3 plot, while running the PE sample and 2) When I removed the FL3 signal out of FL2 while running the 7-AAD tube, I lost all the PE positivity, both in FL1 vs FL2 plot and my final sample (stained with FITC,PE and 7-AAD). I also observed the same phenomenon with APC when I tried to perform 3-color analysis with 7-AAD. Can someone please tell me what am I doing wrong? I've read the papers by Rabinovitch (1986) and also Schmid (1991) but they don't seem to worry at all about compensation problems... Thanks in advance, Dr Ioannis Kotsianidis Clinical Research Fellow Department of Haematology Faculty of Medicine - ICSTM Hammersmith Hospital Du Cane Road LONDON W12 0NN U.K. ********************************************************************** Check out our web site: http://www.cdhb.govt.nz This email and attachments have been scanned for content and viruses and is believed to be clean This email or attachments may contain confidential or legally privileged information intended for the sole use of the addressee(s). Any use, redistribution, disclosure, or reproduction of this message, except as intended, is prohibited. If you received this email in error, please notify the sender and remove all copies of the message, including any attachments. Any views or opinions expressed in this email (unless otherwise stated) may not represent those of Canterbury District Health Board **********************************************************************Received on Mon Jan 31 16:18:00 2005
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