fluorescence compensation (in routine applications)

David Dombkowski dombkowski at helix.mgh.harvard.edu
Fri Jun 3 10:09:31 EST 2005


  My compensation settings are maintained if running AB's with equal 
compensation to last run on instrument. I agree that if you switch 
AB's you must again recalculate or make adjustments after acquiring 
new samples with past settings. I agree with Carol about differences 
among tandem dyes.


  My experience says that a daily procedure to set up compensation is 
not necessary. I have run a a fixed flow cell instrument for many 
years and find the compensation settings at a fixed PMT voltage to be 
identical on a day to day basis. Of course, if instrument is not 
maintained appropriately one should be very concerned about 
compensation settings. I find that even on an instrument such as the 
Vantage SE I can use the same compensation settings daily if 
instrument is maintained appropriately.


It's absolutely necessary, unless you can show that all tandem dyes 
are the same- which they're not!  Every batch is different, and may 
have a very different amount of spillover.  Add to that differences 
in time spent in fixation and exposure to light, and every day is a 
new adventure....

With the use of antibody capture beads, it's so easy- we don't ever 
run an experiment without checking.


On Tuesday, May 31, 2005, at 06:39  AM, Ruud Hulspas wrote:

>I realize that the subject has been discussed numerous times, yet I 
>still	like to bring up the following question.
>In routine applications, where the range in light scatter and 
>autofluorescence of the samples is small, one can set up the flow 
>cytometer with the same PMT settings every day (if nothing has 
>happened to the alignment or performance of the machine). And if the 
>PMT settings are the same, I assume the compensation values are the 
>same (that is, one would have to use high intensity fluorescent 
>samples for each fluorochrome involved during the original 
>compensation procedure in order to avoid under-compensation in case, 
>one day, a sample turns out to be very bright).
>In fact, if it weren't for the irrestistable urge to have 'negative' 
>populations in the first decade of the log scale, one would not have 
>to fiddle with the PMT settings at all, and could maybe measure all 
>samples (even in non-routine applications) without having to change 
>the compensation values on any modern high-dynamic range flow 
>Is a daily procedure to set up compensation values in routine 
>applications really still necessary ?
Ruud Hulspas
David M. Dombkowski
dombkowski at helix.mgh.harvard.edu
Flow Cytometry-Pathology-CNY rm7017
Massachusetts General Hospital-East
149 13th Street
Charlestown, MA 02129
Tel (617)-726-1683
Fax (617)-724-3164

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