Dear Brian, Your problem is likely caused by the sum of several factors. One is spectral cross-talk of 7-AAD into the FL2. This can be troublesome trying to resolve dim expression. You can lower the concentration of 7-AAD at the cost of your CV or use DRAQ-5 as DNA stain alternative. It is excited by 488 nm and has a very large Stokes' shift (see publications of Paul Smith). Virtually no spectral cross-talk into the FL1 and FL2. To solve dim nuclear expression: You might also use FITC instead of PE. Our experience with FITC is that the distribution of cell cycle phase dependent proteins is better resolved than using PE in the same experiments, probably caused by the relative large size of the PE molecule. Furthermore, the possibility to penetrate the nucleus with labelled MoAbs depends on the fixation and permeabilization procedure, especially in the case of using protein cross-linkers (formaldehyde, paraformaldehyde). I don't know what kind of procedure you followed, but in case of a fixative, keep it short (5 min on ice, 0.5 to 1.0% paraform). After fixation you can use methanol, ethanol, lysolecithin or detergents. For nuclear staining, we obtained the best results with methanol. Second, we also observed reduction of FITC as well as PE fluorescence using PI and TO-PRO-3 as DNA stain. (Cytometry 15:117-128, 1994 and Cytometry 28:329-336, 1997). The phenomenon could not be fully explained by absorption or energy transfer. This was very limited as demonstrated by fluorometry experiments using dye mixtures at the lab of Hans Tanke (Sylvius Lab, Leiden). What is causing the effect is still unclear to us but maybe somebody else has a suggestion. Best regards, Willem E. Corver, PhD Department of Pathology Leiden University Medical Centre P.O. Box 9600, Building 1, L1-Q 2300 RC Leiden The Netherlands Tel: +31 71 5266604, Fax: +31 71 5248158 E-mail: W.E.Corver@lumc.nl -----Original Message----- From: Newsom, Brian S. [mailto:BSNEWSOM@txccc.org] Sent: maandag 28 januari 2002 22:02 To: cyto-inbox Subject: 7-AAD/PE problem Question to all of you DNA experts. We have someone who is tring to run an experiment staining a nuclear localized protien with PE and then staining with 7-AAD. The 7-AAD staining works well with a good CV. The antibody staining looks decent (although low percentage and fairly dim) when by itself, but when the 7-AAD is added the antibody staining totally goes away. Is there an energy transfer or steric hinderance issue that may be going on? Any help appreciated. Brian
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