Dear Dr. Abdelhafez You write that the first sample is "highly staining with FITC" and I guess that it is much higher than the control sample used to set compensation. You should not assume that the compensation setting is correct for any sample that is higher intensity than the compensation control. Here is what I think is happening: If your FL1-FL2 compensation is too low at the higher intensity, then the bright FITC will appear positive in FL2. These events are not really PE-positive, so there will be no actual PE signal to overlap into the FL3 channel, but the false PE signal will be subtracted by the FL3-FL2 compensation circuit, causing the negative events to move below the origin of FL3. Regards, Joseph. At 21:07 22/8/2002, 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 non >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 > >Universitätshautklinik Würzburg, AG Kämpgen > >Josef-Schneider-Str. 2 > >97080 Würzburg, 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 -- Joseph Webster, Flow Cytometry Facility Centenary Institute, Sydney, AUSTRALIA. Phone +61-2-9565-6110
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