Re: Sort purity query

Ray

I think your problem is that you don't really have any (or at least 
very many) "positive" cells in P2 (at least as shown in the data 
sample displayed).  The ones shown in the P2 region of the "unsorted" 
stained sample are probably just high outliers of the negative 
distribution.  When you set a region so close to the negative 
population you will sort the high end of the negative population.  At 
the very low fluorescence levels there is a very small difference 
between the "negatives" and the very dim "positives" .	Thus sorting 
this population is expected to give you - upon re-analysis- a 
population which is little different from the starting 
population.  You have simply sorted the high end of a distribution 
which re-distributes back to or near to the original distribution as 
fluorescence level is a statistical measurement dependent on several 
probabilities - e.g. number of photons released from the 
fluorochrome, number of photoelectrons generated from the 
photons.  Thus a small difference in statistics make a big impact on 
the distribution at very low fluorescence levels.  This is very 
different when trying to sort populations which appear only slightly 
different but which say are in the fourth or fifth decade on a log 
scale.	There the differences are likely due to real differences and 
not just statistical distributions - the statistical differences are 
present but only represent a small com[ponent of the "measured" 
fluorescence intensity.  Hope I said this in an comprehensible way.

Larry

At 10:54 AM 12/1/2006, you wrote:
>Hi,
>
>An investigator brought two samples to the lab - one control 
>(unstained cells - '59601.jpeg') and a two color sample 
>('unsorted.jpeg').  Yes, I know, unstained cells aren't the best 
>control, but please overlook that for the moment.
>
>In the attached dot plot ("unsorted"), three regions of interest to 
>sort were identified by the investigator: P2 ('double-stained'), P3 
>(bright PE stained), and P4 (dim PE stained).
>
>We did this as a two way sort.  First, sorting P2-gated cells into 
>the left tube and P3-gated cells into the right tube.	After a 
>period of time, we stopped sorting P2 cells and switched to sorting 
>P4-gated cells into the left tube.
>
>Re-analysis of sorted populations seemed to indicate the P3- and 
>P4-gated sorts had worked reasonably well, but the P2 sorted cells 
>seemed to resemble more the unsorted population.
>
>Could the apparent, poor P2 sort purity result from bleaching of the 
>FITC on these double-stained cells - especially in light of the fact 
>that the PE-stained and sorted populations seemd to fall more 
>closely within their sort gates?  Our 100 mW argon is in the shop 
>again so we are using a Coherent Spectrum 70C laser at 150 mW for 
>excitation of these two colors and maybe that's too much intensity 
>for FITC under the staining conditions of this sample...?
>
>Any thoughts would be appreciated.
>
>Ray Hester
>Univ of South Alabama
>
>
>
>
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Larry W. Arnold, Ph.D.
Research Professor and Director, Flow Cytometry Facility
Department of Microbiology and Immunology
CB# 7290
University of North Carolina
Chapel Hill, NC 27599
Phone: 919-966-1530
FAX: 919-962-8103