Because the "correct" compensation settings on a machine do not correctly compensate the FITC into the FL3 channel. In your case, you have over-compensated this pair... Not your fault, it's the manufacturers who have only provided 2 pairwise compensation settings (FL1 <-> FL2 and FL2 <-> FL3); this does not allow for complete compensation. Depending on your settings, you may have under-compensated fluorescein in the FL3 channel. (For a discussion of this, see my web pages on compensation, particularly, <http://www.drmr.com/compensation/3color.html>). If you are unwilling to use software to completely and properly compensate your 3 color data, you do still have an option that is time consuming and impractical. Set up your machine as you have described below. If you see that the compensation between FL1 and FL3 is off, then lower (or raise) the FL3 voltage until the compensation is off in the other direction about the same amount (i.e., go from too low to too high or vice versa). Then you must go back and re-adjust your compensations for the new voltage settings (the FL2 <-> FL3 comp settings only). Now again check the FL1 vs. FL3 display--you may have to iterate this process a few times. Or, you can just undercompensate all four combinations and use a software package to do it right. (Note that software will not be able to save you in the case you have described below: if any parameter is overcompensated, then you will can't recover the data--once it's on the axis, it's too late.) Good luck mr At 4:07 AM -0700 8/22/02, Dr. Ashraf Abdelhafez wrote: >Hi all: > >O.K. So my samples are ready and I sit at the FACS machine. I setup >the FL1,2 and 3 channel voltages so that the none stained cell >population is in the lower left hand corner. The control stained >cells are nice with only a very small shift if any in the major >population in some of the channels and sporadic events with moderate >staining. I setup compensation properly for the three channels with >cells stained with only single color; the FL3 channel displays a >negative population at the left hand corner and a positive >population down the axis. I put on my first sample that is highly >staining with FITC the cells being dendritic cells and the staining >being for MHCII and the staining in the FL3 channel spreads out and >the negative population goes below the origin of the axis. > >To reiterate, why is it that high FITC staining induces a downward >shift in the FL3 channel although the voltage for FL3 is maintained >at a value set using a non-stained population so that cells are just >above the origin of the axis? How can this be solved? > >Thanks, > >Ashraf Abdelhafez, M.B.B.S. > > > >Dr. Ashraf Abdelhafez > >Universit”tshautklinik W¸rzburg, AG K”mpgen > >Josef-Schneider-Str. 2 > >97080 W¸rzburg, Germany > >Tel. W: +49-931-201-26031 > > H: +49-931-2038-8318 > > Handy (Cellular): +49-931-8090554 > > > >Do You Yahoo!? ><http://rd.yahoo.com/careers/mailsig/new/*http://www.hotjobs.com>HotJobs, >a Yahoo! service - Search Thousands of New Jobs
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