This, of course, would have to be without the addition of PI. Dave ----- Original Message ----- From: "Gerhard Nebe-von-Caron" <Gerhard.Nebe-von-Caron@unilever.com> To: cyto-inbox <cytometry@flowcyt.cyto.purdue.edu> Sent: Friday, February 01, 2002 9:29 AM Subject: RE: 7-AAD/PE problem > > Apart from energy transfer consider electron / photon overflow and compensation > problems. > > Excitation of TP3 with a HENE LASER does give you a nice TOPRO signal but > should not prevent FRET in the blue laser beam if there is still an energy > transferring dye combination present. > > > Regards > > Gerhard Nebe-von-Caron > > Research Scientist > Applied Science & Technology Group > SEAC - Safety and Environmental Assurance Centre > Unilever Colworth, Sharnbrook, Bedfordshire, UK - MK44 1LQ > > Tel: +44 (0)1234 264822, Fax: +44 (0)1234 222552 > E- mailto:Gerhard.Nebe-von-Caron@unilever.com > > > > > -----Original Message----- > From: DAVID M CODER [SMTP:d_coder@msn.com] > Sent: Wednesday, January 30, 2002 10:20 PM > To: Cytometry Mailing List > 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 > > > > > > Dear Brian, > > > > [part deleted] > > 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. > 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 > > > >
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