From: Tim Denning (tdenning@liai.org)
Date: Fri Sep 27 2002 - 12:21:57 EST
Dear Group, I am not very familiar with CFSE analysis, much less with 3 additional colors, so I was hoping someone might offer me some help. Thank you very much for your time and help in advance. Can anyone help me with instrument setting for 4-color with CFSE on the FACSCaliber? I am interested in general voltages and compensation to make sure I am in the right neighborhood (currently I think I am not even in the right country so-to-speak). The person showing me how to do this is labeling cells with >5uM CFSE (the more/greener the merrier method?) and during analysis this person is greatly increasing the FL1 voltage and then compensating 99.9% between FL1 to FL2 and quite high between other channels as well. The person doing this tells me that using less CFSE is not good b/c the peaks will not be "tight" and I will not be able to see all the divisions clearly. Since I am a novice at this I understand his reasoning? This person is incredibly bright and really knows his science, so I can't imagine he doesn't have a reason for his method, maybe I just can't understand it myself. I am attempting to simultaneously stain CFSE-labeled cells with PE, PE-Cy5, and APC and I not having much luck. My experimental set-up is labeling freshly isolated thymocytes with CFSE, expanding them with various cytokines in vitro for 4 days during which time they clearly undergo 3-5 divisions. I understand that my CFSE concentration (>5 uM) may be WAY too high and I am working on trying various dilutions 2.5, 1, 0.5, 0.1 uM on my own. So here are my questions: 1. Is there a preferred dilution for in vitro studies and with that dilution what are the general (I don't need exact numbers, just ballpark) voltages and compensation values I might expect to use? 2. Is there a trick to get those beautiful, sharp peaks I sometimes see in the literature? Much thanks to the group and my apologies if my questions are repetative and/or sophmoric. Tim Timothy L. Denning, Ph.D. La Jolla Institute for Allergy and Immunology Division of Developmental Immunology 10355 Science Center Drive San Diego, Ca 92121 (858) 678-4534 (tel) (858) 678-4595 (fax) tdenning@liai.org http://www.liai.org
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