RE: Sort purity query

Hi Ray

It helps to embed multiple pictures into one word document to make it a
bit easier to compare them.

It looks to me as if you sort the cells on the right side of a normal
noise distribution. The noise here is partially down to compensation
probably including an offset? I would expect them to come back in the
middle of the Gaussian population. Pity the PE only or FITC only stain
do not exist.

Regards

Gerhard



> -----Original Message-----
> From: Ray Hester [mailto:rhester@jaguar1.usouthal.edu] 
> Sent: 01 December 2006 15:54
> To: Cytometry Mailing List
> Subject: Sort purity query
> 
> 
> 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
> 
> 
> 
>