My name is Sian Rizzo and I am a student at St.George's Hospital Medical School in London (UK). I am also interested in using 7AAD for cell cycle analysis in conjunction with surface antibodies (FITC and PE). I've been using PI but the compensation is proving problematic, to get the signal at the recommended channel my volts are too high to compensate out of FL3. Also having FL3 as the cell cycle parameter is better for me due to availability of directly-conjugated antibodies. I have just completed an experiment where I labelled suface antigens, then used a Dako fix+perm kit followed by 7AAD. I had a good peak with the 7AAD for G0/G1 but there was no distinct G2/M peak, though this may be the case for the cells I was using (unlikely to be zero though). I used ModFit LT for the cell cycle analysis. I came across a reference that cited histone interference with 7AAD binding, is this crucial? Do I have to do a histone digest? Also, 7AAD is supposed to bind specifically to DNA, would anyone still recommend an RNase step (as for PI)? Thanks in advance for your help! > My name is Kate Dugan and I am a graduate student in Dr. > Tim Manser's lab at Thomas Jefferson University. I want to > do a cell cycle analysis using CFSE, PE, and 7-AAD. I > tried this one time but without success because of the > 7-AAD staining. I was wondering if you had any suggestions > for using 7-AAD in this application. I will greatly > appreciate any information that you could share with me. > Thank you for your time. ----------------------------------- sian rizzo @sghms.ac.uk St. Georges Hospital Medical School Opinions expressed those of the author and not the institution
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