Maris As long as you have the lasers together you can not resolve the problem except by compensation (this is the problem in the Coulter machines because even thought the lasers can be spatially resolved the emissions from separate lasers are not focused through pinholes). If you separate the lasers and use a Vantage or MoFlo you will solve the problem (mostly on the MoFlo probably completely on the Vantage) without a need for compensation because the emission from each laser is focused through spatially resolved pinholes and thus in theory the HO emission will not get to the APC detector. For the most part this works in spite of worries about light piping. The emission from HO33342 at this wavelength is only a small percentage of the total HO emission, however, since the total emission from the HO is very large the HO670 emission relative to an antibody-APC stain (ie. the HO is very bright)can be a problem. This is a much bigger problem in the FITC channels since the HO emission at this wavelength is much more than at the 670nm range. Some people think you should be able to solve the problem with filters but that only displays a flaw in their thinking since there is really HO emission across a wide wavelength range. Larry At 03:15 PM 1/11/2002, you wrote: > Reply to: Re: HO33342 into 670/20 filter??? >Hi Larry, >Thanks for your help. HO33342 does emit at that wavelength? That would >explain it. It was very easy to compensate (nothing extreme), so I'll >stop worrying about it, and go forward with the experiments. >We ran the experiment on a FACS Vantage (so the UV and 647 excitation were >coaxial). Any suggestions? Next week we will try it (again) on the >Vantage, and on a Mo-Flo as well to see if changing the instrument makes a >difference. > >Thanks again, >Maris >Larry Arnold wrote: > >Maris > > > >What instrument are you using? HO33342 does emit significantly at > that >wavelength so you can get cross-talk between lasers. After I know > what >instrument you are using I may be able to suggest something. > > > >Larry > > > >At 11:12 AM 1/10/2002, you wrote: > > > >>Hi Everyone, > >>Happy New Year to All! > >> > >>I am at a loss to explain why cells that are stained with Hoechst33342 > are >>increasing > >>the background in my APC signal...HELP! We were looking at DNA > using >>HO33342 (excited by > >>UV, emission through a 450/20 filter) and APC (surface receptor, > using >>647nm excitation > >>and emission through a 670/20 filter). There was nothing out of > the >>ordinary visible > >>during the alignment (no laser noise increasing the background of > either >>signal, etc). > >>We set the APC negative control within the first decade, looked at > the >>APC+ control, and > >>saw a distinct positive population. When we put on a sample that > was >>stained with HO > >>(APC-), the signal in the APC channel went into the higher end of > the >>second decade. > >>At first, we were splitting the two signals with a 510LP, and > after >>witnessing the > >>shift in background, we switched to a 640LP...we still saw the same shift. > >>We compensated the HO out of the APC signal, and the experiment seemed > to >>work as > >>expected (read: the researcher was happy with his results), but > does >>anyone have any > >>idea what happened? I've been looking at every spectrum figure I > can >>find, and have > >>looked at the filter specs and I can't find any reason for > that >>shift. Any advice, > >>as always, is sincerely appreciated. Thanks, > >>Maris > >> > >>Maris A. Handley > >>Dana-Farber Cancer Institute > >>J415 > >>44 Binney St. > >>Boston, MA 02115 > >>(617)632-3139 > >>maris_handley@dfci.harvard.edu > > > >Larry W. Arnold, Ph.D. > >Associate Professor > >Director, Flow Cytometry Facility > >Department of Microbiology and Immunology > >Lineberger Comprehensive Cancer Center > >CB# 7290 > >University of North Carolina at Chapel Hill > >Chapel Hill, NC 27599 > >Phone: 919-966-1530 > >FAX: 919-962-8103 > > > >RFC822 header > >----------------------------------- > > > > Received: from europa.dfci.harvard.edu ([155.52.44.41]) > by >PHSEXCHICI2.Partners.org with SMTP (Microsoft Exchange Internet Mail > Service Version >5.5.2650.21) > > id CT5XLRV9; Fri, 11 Jan 2002 15:57:18 -0500 > > Received: from redtail.med.unc.edu (redtail.med.unc.edu [152.19.4.7]) > > by europa.dfci.harvard.edu (8.10.2+Sun/8.11.3) with ESMTP id > g0BKtq600444 > > for <Maris_Handley@dfci.harvard.edu>; Fri, 11 Jan 2002 15:55:52 > -0500 (EST) > > Received: from PC231H602A.med.unc.edu (pc231h602a.med.unc.edu > [152.19.43.97]) > > by redtail.med.unc.edu (8.9.3/8.9.3) with ESMTP id PAA19243; > > Fri, 11 Jan 2002 15:57:00 -0500 (EST) > > Message-Id: <5.0.2.1.2.20020111161015.020dd388@imap-ns.med.unc.edu> > > X-Sender: lwarma@imap-ns.med.unc.edu > > X-Mailer: QUALCOMM Windows Eudora Version 5.0.2 > > Date: Fri, 11 Jan 2002 16:12:20 -0500 > > To: Maris Handley <Maris_Handley@dfci.harvard.edu>, > > Cytometry Mailing List <cytometry@flowcyt.cyto.purdue.edu> > > From: Larry Arnold <lwarma@med.unc.edu> > > Subject: Re: HO33342 into 670/20 filter??? > > In-Reply-To: <200201101711.MAA18384@flowcyt.cyto.purdue.edu> > > Mime-Version: 1.0 > > Content-Type: text/plain; charset="us-ascii"; format=flowed > > Larry W. Arnold, Ph.D. Associate Professor Director, Flow Cytometry Facility Department of Microbiology and Immunology Lineberger Comprehensive Cancer Center CB# 7290 University of North Carolina at Chapel Hill Chapel Hill, NC 27599 Phone: 919-966-1530 FAX: 919-962-8103
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