From: John Altman (altman@microbio.emory.edu)
Date: Wed Sep 10 2003 - 10:23:25 EST
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 PS - I've only just begun the website, and I plan on putting up lots of more useful stuff in the coming months. -- 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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