Well I nearly went ballistic when I read this too ....and I just left it.......but since Howard opened the door..... About 13 or 14 years ago, any manuscript that was sent to me for review that used figures with FL1, FL2. etc. was returned to the editor unreviewed and identified as not acceptable for publication. Now an associate editor of Cytometry, I won't even send a paper out for review if the author uses nonsensical labels. There is no place for a discussion of FL1, fl5 OR fl15... We banned the use of these label in Current Protocols in Cytometry when we started CPC in 1996. Cytometry is a science - as such, we should expect carefully documented technical notes. That includes references to the nature of the signal being described. I suppose it is important for is to constantly remind people of these issues, but they are not minor issues when one is trying to work out what exactly is being reported on. Kind regards Paul Robinson Howard Shapiro wrote: > James Marvin wrote: >> I've noticed this also. I chalked it up to residual DAPI in the >> sample line for a while but im not sure thats really what it was. And >> your right, its didnt happen all the time, and i was running lots of >> fixed samples so thats why i thought it was DAPI. I think that I >> watched the Violet 1/FL-6 fluorescence increase as the sample went >> through, another reason i thought it was dapi. I believe it would pop >> up in the FL-7/violet-2/pacific orange detector also. >> >> Let me know what you come up with >> >> thanks >> J >> >> At 03:06 PM 3/11/2008, Kerry Lavender wrote: >>> Hello Flow-ers, >>> >>> I wonder if anyone has ever experienced or has a solution to this >>> problem? >>> >>> We have been running 8 colour panels on a Cyan machine for about 12 >>> months now with no problems. Suddenly, and with no particular pattern to >>> it we are occasionally seeing HUGE and BIZARRE >>> autofluorescence/non-specific staining ONLY in the FL-6 channel (and >>> primarily affecting the negative population). >>> >>> It has happened with 3 separate Pacific Blue conjugates now, 2 >>> extracellular markers (CD3 PB and Vb11 BT+ Strep Pacific Blue) and one >>> intracellular marker (TNF PB). And in a number of staining panels >>> ranging from 4-8 colours. Sometimes it manifests as 3 populations >>> shifted up along the PB-axis even if the sample is negative or contains >>> no pacific blue antibody. >>> >>> We have tested the possibility of it being due to old formaldehyde >>> containing solutions but do not see any improvement with new ones. We >>> have also ascertained that it is not an issue with our specific machine >>> as it reproduced on a second machine. There appears to be no rhyme or >>> reason to when it occurs in regard to stimulated vs non-stimulated >>> samples, permeabilization, presence of protein transport inhibitors or >>> length of time sitting in fixation buffer. >>> >>> We are really at a loss - can anyone help?? > I am not suggesting a solution to the problem posed, but pointing out > another, potentially serious, problem. > > I find it difficult to believe that, with the profusion of flow > cytometers with as many as five working laser beams and 18 fluorescence > measurement channels, "FL-6" will be the same for all of them. If you > are measuring fluorescence, you need to specify the excitation > wavelength (usually easy, because it is a laser line) and the emission > region, defined for bandpass filters by center wavelength and bandwidth > (e.g., 520/30) or for long or short pass filters by cut-on or cut-off > wavelength (e.g., 665 LP). Yes, I know, FL1, FL2, and FL3 are pretty > standard in instruments with 488 nm excitation, but the standard that we > in ISAC would like to impose for publication will get rid of them, too, > in favor of descriptions that don't require familiarity with any > particular kind of cytometer to understand. I have run across a number > of situations in which people couldn't get the same results because they > didn't realize they were using different filters for "FL4" or whatever, > and the more choices we have for excitation wavelengths and measurement > regions, the more often this will happen, unless we become more precise > in our characterization of measurement channels. > > -Howard > -- J. Paul Robinson SVM Professor of Cytomics Professor of Immunopharmacology & Biomedical Engineering Director, Purdue University Cytometry Laboratories President, International Society for Analytical Cytology Purdue University Cytometry Laboratories Bindley Bioscience Center 1203 West State Street Discovery Park, Purdue University West Lafayette, IN 47907-2057 Ph (765) 494 0757; Fax (765) 494 0517 email: jpr@flowcyt.cyto.purdue.edu www.cyto.purdue.edu Join ISAC - www.isac-net.org Change lives today - www.cytometryforlife.orgReceived on Tue Mar 18 11:38:00 2008
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