Post Sort Quenching -Reply

Antony Bakke bakkea at ohsu.edu
Sun Feb 9 13:02:27 EST 1997


O. Joseph Trask, Jr, writes:

I would like to find out what the consensus is on the loss of fluorescence
of post sorted viable cells during re-analysis....  Is it normal to adjust the
pre-sorted region (within a few
channels) to check the purity of the sort OR does one leave the region in
the exact position from the original sort equation? 

--------------------------------------------

I certainly see a slight loss of fluorescence after sorting.  I believe this is
a combination of quenching of the fluorochrome and modulation of the antigen and
antibody off the cells.  I usually adjust the cursors for reanalysis after
sorting a few channels to allow for this.  The adjustment is empirical based on
the pattern and I am certain to exclude clearly negative cells.  In addition, I
usually set the threshold higher during sorting so the instrument is not
spending time analyzing debris, red cells, dead cells, etc.  Therefore, some of
these particles may have been sorted into your "pure" populations in adjacent
drops because they were not detected and did not trigger an abort.  It is
important to exclude this debris and dead cells from the reanalysis because the
sorter never had a chance to exclude them originally.

Good Luck,
Tony Bakke, PhD
Director, Clinical Immunology and Flow Cytometry
Dept of Pathology
Oregon Health Sciences University
Portland, OR
bakkea at ohsu,edu



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