Jake Jacobberger is correct. Most likely the phenomenon reflects rapid diffusion of the dye and/or ions from the core sample stream to the sheath stream when they meet upstream in the flow channel. The diffusion leads to a decrease of dye concentration in the sample (core) stream which breaks the equilibrium between the dye and its binding sites in the cell. The changeable staining pattern is observed until new equilibrium establishes which takes some time of flow run. We observed this phenomenon using acridine orange, the dye that is extremely sensitive with respect to even minute change in its concentration or concentration of counterions such as sodium or divalent ions in the sample stream. The phenomenon is additionally exacerbated in instruments that have long sample lines such as old Ortho instruments and can be diminished by faster flow rate. We underscored this in our old papers describing the use of acridine orange (e.g. Darzynkiewicz, Z.: Simultaneous Analysis of Cellular RNA and DNA Content. In: Methods in Cell Biology, Flow Cytometry (2nd edition). Z. Darzynkiewicz, J.P. Robinson and H.A. Crissman (eds.), Academic Press, New York, N.Y. 1994, pp. 401-420, see pages 411-412.) I wish Merry Christmas, Happy Holidays, and the very best in the New Year to all FLOWERS, Zbigniew Darzynkiewicz, M.D., Ph.D. Professor of Pathology and Medicine Director, Brander Cancer Research Institute New York Medical College BSB, Room 438 Valhalla, N.Y. 10595 www.darzynkiewicz.com/zbigniew/ -----Original Message----- From: James W Jacobberger [mailto:jwj@case.edu] Sent: Thursday, December 20, 2007 4:09 PM To: cyto-inbox Subject: Re: 7AAD and PY for G0 and G1 - signal fluctuation at beginning of analysis 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/d932f9e462b4bfa1e5928ddde8237d7b83bc 4e4c.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/729275a18fc1ace22c3c231d8f7cc32536b0 e86e.ppt >> > > James Marvin > Manager, RHLCCC Flow Cytometry Facility > http://www.basic.northwestern.edu/sharedresources/flowcytometry/flowcyom etrysite.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 Sat Dec 22 14:58:00 2007
This archive was generated by hypermail 2.1.8 : Wed Jan 31 2007 - 03:12:00 EST
