Re: Sort purity query

Dear Ray

I doubt photobleaching plays a part here. I think your P2 gate contains the
upper end of the FITC channel negatives which is just noise and not FITC
staining at all. Cells from the the P2 gate are the same as the cells to the
left of the P2 gate hence the similarity to unsorted cells. Maybe your customer
forgot to add the Fitc antibody! Or the events are so rare that they are
totally swamped by the negatives, either way its the investigators fault.

Best

Simon


 Quoting Ray Hester <rhester@jaguar1.usouthal.edu>:

> 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
>
>
>


-- 
Simon Monard
Cytometry Lab Manager
IBMC
Porto
Portugal

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