From: John Altman (altman@microbio.emory.edu)
Date: Wed Sep 10 2003 - 21:59:48 EST
Dave, Actually, I would argue that the fact that we see the events on the blue laser (at least for the lower wavelengths) but not the violet (not UV- that was my mistake) and red lasers suggests that it was not debris. I didn't show the scatter plots, but the data in the histograms was relatively tightly gated on a distinct lymphocyte population. On the other hand, your comments in the first paragraph seem to me as if they ought to be correct, but I think that somehow it's still more complicated than this. I suspect that most of the explanation was discussed in Marty Bigos's posting about on-axis events: http://www.cyto.purdue.edu/hmarchiv/current/0158.htm I also heard today from a BD Technical Applications Specialist that they are aware of the difficulty of setting proper PMTs for regions of the excitation and emission spectra where there is little cellular autofluorescence. If I hear anything concrete, I'll report back. John On Wednesday, September 10, 2003, at 05:30 PM, David Novo wrote: > Hi John, > > If you say that the operator cranked down the gains of the PMTs so > that the unstained cells were in the first quadrant, well that is > obviously not the case if what you are showing are really the cells. > Because you can see that for the UV and HeNe laser he/she turned them > down so low that half of the cells were on the axis. > > Maybe when he turned down the PMTs he/she gated on cells and when you > are showing the data, you are showing all the events, but the stuff on > the axes is debris. It makes sense that it is debris, because I don't > see why there should be a bimodal distribution of unstained data in > the HeNe and Violet, if all the events you are showing are really > cells. > > -Dave > > > > -------Original Message------- > > From: John Altman > Date: Wednesday, September 10, 2003 1:17:50 PM > To: Cytometry Mailing List > Subject: Help us on PMT settings on the Aria > > At the risk of revealing what a fool am I, I ask your help in setting > the proper voltages on our new FACS Aria. > > Last week, I asked one of my techs to perform a titration of an > Alexa-430-labeled anti-CD8 (we made it ourselves) and to acquire the > data on a Calibur and the Aria. The tech did the flow work on the > Calibur himself, and handed the samples off to our Aria operator, > without providing the operator with much information. > > The Aria operator tells me that he set the PMTs for each channel on the > Aria by cranking them up higher than they should be, and then backing > them down so that the cells filled the lowest decade. He performed this > on an unstained sample of whole human blood (we don't often use isotype > controls, but I don't want to get into that debate unless forced to). > All of this seems sensible to me. > > The problem is that we got data out that looks like this: > > Link to data: > > http://www.microbiology.emory.edu/altman/f_protocols/f_flowCytometry/ > HelpPMT.html > > As you can see, in several of the channels (Violet1-A, Violet2-A, > PE-Cy7-A, APC-A, APC-Cy7A), we have most of the cells slammed against > the axis, yet the on scale data spans nearly two decades. > > How do we solve this "problem"? Do we simply run at higher voltage > settings on the PMTs? > > Thanks, > > John -- John D. Altman Associate Professor Emory Vaccine Research Center 954 Gatewood Road Atlanta, GA 30329 altman@microbio.emory.edu http://microbiology.emory.edu/altman Office Phone: (404) 727-5981 Lab Phone: (404) 727-8914 FAX: (404) 727-8199 Assistant Phone: (404) 727-8778
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