James, There is a paper on this published in Cytometry in the early eighties. Although I suppose it is not the only possible reason, most likely, it is mixing of sheath and sample that causes the change in fluorescence and what you see is the time for the machine to come to stable hydrodynamic focusing. happy holidays, jake James Marvin wrote: > Ive noticed the same thing with a Zinc binding dye on our cyan. In > this case the fluorescence steadily drops for the first........10-15 > seconds and then is perfectly stable. I'm usually a little skeptical > when doing any type of analysis where Median or mean is gonna be > important so i just happened to be looking at a plot ofTime vs > fluorescence. > > j > > > > > At 07:01 AM 12/19/2007, Roberts Joanna wrote: >> >> >> Dear Flow people, >> >> >> >> As always, i am so glad that I can direct my questions to a bunch of >> people with lots of good ideas. >> >> >> >> I have a customer doing an experiment on a cell line looking at cell >> cycle and G0/G1 using 7AAD and PY. The experiment is being run on a >> CyAn. The problem we are seeing is that for the first few seconds of >> data acquisition, the signals start low and increase to a stable level. >> This seems unexpected. Has anyone seen anything like this? The early >> parts of each measurement have to be discarded. It seems that these >> signals should be stable throughout and not fluctuate. >> >> >> >> Here are some other details of experiment set up >> >> - using low flow rate to get good CVs (at this flow rate, there >> is a delay of about 30 secs between placing cells on the machine and >> seeing data) >> >> - measuring linear area signals >> >> - triggering on 7AAD channel (but if we use FS we still see the >> same thing) >> >> >> >> What seems really weird to me is that >> >> - running the same sample over and over again gives the same >> result each time (low signals quickly climbing to a stable level) SEE >> TOP LINE OF GRAPHS IN ATTACHMENT >> >> - this effect is not at all noticeable when running alignment >> beads BOTTOM LINE OF GRAPHS IN ATTACHMENT >> >> - if you stop acquisition, leave tube on machine, scratch your >> head, and then start acquisition again, the signals are STABLE (no >> fluctuation) >> >> - effect is most obvious in PE channel (for PY) but is also >> visible in the PECy5 channel (7AAD) >> >> >> >> Because I don't see this effect with the alignment beads, I find it hard >> to believe it is the machine but then I can't think of an explanation in >> biological or physical terms either. Can anyone help? >> >> >> >> I will be delighted to hear any thoughts about this. >> >> >> >> Best wishes, >> >> Joanna >> >> >> >> >> >> Flow Cytometry Core Facility >> >> EPFL, Swiss Federal Institute of Technology >> >> SV-SG, Station 15 >> >> CH 1015, Lausanne >> >> Switzerland >> >> >> >> +41 21 69 39 547 >> >> >> >> >> Content-Type: text/plain; name="warning1.txt" >> Content-Disposition: inline; filename="warning1.txt" >> Content-Transfer-Encoding: 7bit >> MIME-Version: 1.0 >> X-Mailer: MIME-tools 5.411 (Entity 5.404) >> >> This attachment - 'image001.gif' - 862 Bytes - can be viewed at >> http://www.cyto.purdue.edu/MD-parts/d932f9e462b4bfa1e5928ddde8237d7b83bc4e4c.gif >> >> >> >> Content-Type: text/plain; name="warning2.txt" >> Content-Disposition: inline; filename="warning2.txt" >> Content-Transfer-Encoding: 7bit >> MIME-Version: 1.0 >> X-Mailer: MIME-tools 5.411 (Entity 5.404) >> >> This attachment - '18.12.7 7AAD and PY.ppt' - 174.59 KBytes - can >> be viewed at >> http://www.cyto.purdue.edu/MD-parts/729275a18fc1ace22c3c231d8f7cc32536b0e86e.ppt >> > > James Marvin > Manager, RHLCCC Flow Cytometry Facility > http://www.basic.northwestern.edu/sharedresources/flowcytometry/flowcyometrysite.html > > 312-503-0913 (office) > 312-908-1294 (lab) > > > "What must a man possess who possesses the Possessor of all things?" > Savanarola > > -- James W Jacobberger, PhD Professor of Oncology, Associate Director Shared Resources, Director Cytometry Core, Case Comprehensive Cancer Center Case Western Reserve University 10900 Euclid Avenue Clevland, OH 44106- Phone: 216-368-4645 FAX: 216-368-8919Received on Fri Dec 21 12:38:00 2007
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