I'd recommend that you try lower concentrations of the CFSE. I've tested the entire range from 0.5 uM to 25 uM, and anything greater than 0.5 uM produces too much cross-over onto FL-2 to compensate for. This will of course depend on the length of time that you passage your cells (i.e. division cycles) between the time you label with the CFSE and the time you read them on the cytometer. Ryan. _/ \__/ \__/ \__/ \__/ \__/ \__/ \__/rhung@vcn.bc.ca__/ \__/ \__/ \_Apoptosis=programmed cell death/ \__/ \rwhung@interchange.ubc.ca_/ \__ _/ --you can't live without it!/ \__/ \__/ \__/ \__/ \__/ \__/ \__/ \__/ \__/ \__/ \__/ \__/ \__/ \__/ \My words Copyright (C) 2001 \__ On Thu, 15 Nov 2001, Andrea Dewar wrote: > > 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 suggestions 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 - 19:01:40 EST