It is my understanding that the "tightness" of the peaks is a direct reflection of the uniformity of the labelling. I believe this has less to do with the concentration used (assuming it is sufficient to label all the cells) than with the technique for mixing CFSE with the cells and by the uniformity of cell volume of the population being labelled. With this in mind you can try various things to improve the labelling. For example, there are some helpful suggestions from Chris Parish (the senior author on the original CFSE division paper) in his article in Current Protocols in Immunology. PARISH, C., and WARREN, H. S. (2000). Use of the Intracellular Fluorescent Dye 5-(and -6)-carboxyfluorescein diacetate succinimidyl ester (CFSE) to Monitor Lymphocyte Migration and Proliferation. In "Current Protocols in Immunology". Wiley and Sons. I don't usually do CFSE on Calibur (mainly because I usually need more than 4 colours for my CFSE experiments) . But I do know that I and other routinely have ~20-40% compensation between FL1 and FL2. (BTW I think 99.9% is the maximum that CellQuest will allow - in that case how does he know he doesn't actually need more than 99.9?) Has your colleague ever assessed the level of toxicity of the concentrations he is using? In my experience CFSE can be quite nasty in high concentrations. I hope that helps. Adrian ______________________________________________ Adrian Smith (Research Officer) T CELL BIOLOGY GROUP Centenary Institute of Cancer Medicine & Cell Biology Locked Bag No.6 Newtown, NSW 2042 AUSTRALIA. Ph: (02) 9565-6198 Fax: (02)-9565-6103 >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 > >
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