Re: Sort purity query

Ray,

The explanation is that you have no genuine fluorescein signal in the  
FITC-A channel; just the normal, random background distribution  
contributed to by electronic noise, leaking laser light, compensation  
calculation error distribution, cosmic rays, bad vibes, etc..  The  
cells within this distribution that happen to fall into the P2 region  
during sorting will, when re-analysed appear randomly placed across  
the original FITC-A background distribution (as the data shows).  You  
can see the unstained sample also has cells registering higher in the  
FITC-A distribution than the lower FITC-A limit of P2.

Frank Battye.

On 02/12/2006, at 2:54 AM, Ray Hester wrote:

> 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...?

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