Simon - In addition, there is another factor to consider, photon statistics. Depending on your sample, some, or all, of the "contamination" you see may be due to the statistical variation in the measurement. For example, if you take a nice Gaussian bead peak, sort the upper shoulder, and reanalyze, you will see the entire peak. The same is generally true if you were to sort the upper edge of an autofluorescent cell population. In your case, the "weak shoulder" could be a convolution of autofluorescent cells and weakly staining cells. When rerun after sorting, the autofluorescent cells would recapitulate the autofluorescent population and be seen "out of window". Moreover, as with the beads, the weakly staining cells would also have a distribution which probably extends below your gate. Marty >Hi Simon, > >I have had the same problem you described and I discussed it with BD's >sorter specialists. >Various points have to be taken into account: >Weak fluorescence can result from low antigen density, bad >protein/fluorochrom ratio or insufficient amount of used antibody. Even the >antigen distribution or the fluorochromes distribution (patches) when >labelling cell membranes can give slightly different signals when passing >the analyzing point. The fluorochrome itself (tandemconjugates or not) can >give weaker or stronger signals. >If a sort gate is set all cells or particles which fit these criteria will >be sorted if they can be sorted (no coincidence). When passing the laser >light, weak fluorecent cells may loose fluorescence by bleaching. Therefore >reanalyses often show the sorted cells not fit the sort gate again. And if >the desired population was close to the negative unwanted ones it is >possible that during reanalyses the fluorescence of the sorted cells fall >into the negatives. >The only argument is loss of fluorescence during the sort. > >RegardsVolker > >Volker Eckstein PhD >Dept. of Internal Medicine V >Medical School of the University >University of Heidelberg >Heidelberg - GERMANY >volker_eckstein@med.uni-heidelberg.de > > > >-----Ursprüngliche Nachricht----- >Von: Simon Monard [ mailto:smonard@trudeauinstitute.org ><mailto:smonard@trudeauinstitute.org> ] >Gesendet am: Montag, 29. Oktober 2001 20:22 >An: Cytometry Mailing List >Betreff: Sort purity > > >Hi folks. >Can anyone point me to a discussion of ways to estimate the purity of a >sorted population >of cells. This is clearly easy when you are sorting very bright cells from >negatives >but more problematic when sorting cells from a "shoulder". I remember seeing >a nice >discussion on this subject but cannot remember where. I've been sorting some >very >weakly positive cells from a population, these cells post sort overlap the >negative >cells quite a bit. My customer seems unhappy about that. >Thanks > >Simon Monard >FACS Lab Manager >Trudeau Institute >Saranac Lake >NY12983 > >Ph 518 891 3080 X352 -- Marty Bigos Director, Flow Core 415-695-3832 Gladstone Institute of Virology and Immunology Building 3 SFGH
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