Andrea, You're running these samples on a Coulter XL . . . and that "problem" you're seeing is unique (as far as I know) to the XL. (I have 2 Elite cell sorters, running the old DOS Elite acquisition/analysis software -- they don't have this problem . . .) When compensating, many of the negatives will bury against the baseline . . . a situation even Coulter recognizes as problematic, as they now include an option in their (ACS's) EXPO32 software to positively "offset" the negatives, placing them in the first decade . . . and in view. So, EXPO 32 is the only way to eliminate this "artifact." MAK. >>> Andrea Dewar <andrea.dewar@imvs.sa.gov.au> 11/15/01 19:28 PM >>> Hi there. I'm having a big problem with compensating cells double stained with CFSE (FITC) and PE on our Coulter flow cytometer. On a dot-plot, to bring single stained CFSE cells into the 1st decade of the scale, 75% or more of my cells slam against the axis before the remaining 25% of cells are pulled into the first decade region. When my cells are counter stained with a PE antibody, the majority of negative cells are still slammed onto the axis, so that if you don't look closely, you can't see these cells and all events appear to be double positive (whereas at least 25% of the cells are actually negative). This is a problem specific to the Coulter machine as the same cells were run on a BD machine. The cells did not slam into the axis when compensated on the BD machine. The level of compensation used on the Coulter machine is not high (28.1) and the problem is not becuase the cells are stained too brightly (the problem still occurs when the cells are sitting at 2000). Any uggestions would be thoroughly appreciated. Andrea Dewar PhD Student Institute of Medical and Veterinary Science Adelaide South Australia AUSTRALIA
This archive was generated by hypermail 2b29 : Sun Jan 05 2003 - 19:01:39 EST