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@ohsu,edu
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