-----Original Message----- From: DAVID M CODER [mailto:d_coder@msn.com] Sent: woensdag 30 januari 2002 23:20 To: cyto-inbox Subject: Re: 7-AAD/PE problem ----- Original Message ----- From: "Corver, W.E. (PATH)" <W.E.Corver@lumc.nl> To: cyto-inbox Sent: Tuesday, January 29, 2002 9:06 AM Subject: RE: 7-AAD/PE problem David and Brian wrote: > Energy transfer seems the best explanation. FITC and PI are a near optimal pair; PE and PI less so, but still there is overlap. PI and TO-PRO-3 are a very good pair: the emission of PI overlaps well the absorption of TO-PRO-3 (e.g., a HeNe laser at 633nm excites TP-3 very well), and both dyes intercalate into DS-DNA. In fact, the only way to observe TO-PRO-3 fluorescence when only a 488nm line is available is by transfer from PI. All such energy transfer (FITC, PE --> PI; PI-->TP3) is avoided with direct excitation of TP-3 by red (633 to 647nm) excitation. Best regards, Dave ---------------- David M. Coder, Ph.D. Consultant in Cytometry email: d_coder@msn.com tel./messages: 206-499-3446 > -----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 > Dear David, Indeed PI and TO-PRO-3 are excellent candidates for FRET, as we and several others have published. Also, FRET might be involved in this case. However, we are still puzzled. We observed a significant reduction in cell surface fluorescence (FITC and PE) after adding PI at high concentrations (50 µg/ml) using viable cells. Using TO-PRO-3 (lower concentrations, 2 µM) we obtained comparable results. We could not explain this quenching of fluorescence due to absorption (unlikely due to the law of Lambert-Behr) nor FRET (we did some measurements). Especially the effect of TO-PRO-3 on FITC is intriguing due to large differences in exc./emis. wavelengths of the dyes. Maybe Howard has an idea. 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
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