I agree that the apparent reduction of FITC signal (and not PE) for cytoplasmic proteins is not at all clear. (Were the experiments repeated using only TO-PRO-3 and a red laser where PI crosstalk is eliminated?) For nuclear antigens in close proximity to DNA, energy transfer would be expected for the cases note below. p53 can exist in both cytoplasmic and nuclear compartments, so one would expect differences related to the position of a cell in the cell cycle. Dave ---------------- David M. Coder, Ph.D. Consultant in Cytometry email: d_coder@msn.com tel./messages: 206-499-3446 ----- Original Message ----- From: "Corver, W.E. (PATH)" <W.E.Corver@lumc.nl> To: cyto-inbox Sent: Thursday, January 31, 2002 6:14 AM 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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