Re: Sort purity query

Hi Ray,

My hunch is that the P2 region contains doublets that fall apart when 
re-analysed (or were just coincident in the fist place and not necessarily 
stuck together.  It might be worth checking pulse width v height or area as 
well as gating more stringently on FSC (or looking to see if the P2 cells 
correlate with greater FSC or pulse width)

cheers

Ray

ps if you've not managed to get some of the longer 4 way sort tube holders 
yet, I've got a couple I could spare - though I tend to just drop a spacer 
in the bottom of the short tube holders (a ball of foil in fact) to elevate 
the outer two tubes when needed - it's easier than changing the holders 
over.


----- Original Message ----- 
From: "Ray Hester" <rhester@jaguar1.usouthal.edu>
To: cyto-inbox
Sent: Friday, December 01, 2006 3:54 PM
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
>
>
>


--------------------------------------------------------------------------------


> This attachment - 'DOTPLOT_59601.jpeg' -  25.60 KBytes - can be viewed at
> http://www.cyto.purdue.edu/MD-parts/f9352720fa4f27c3a9af098e34674e6ae6c64ef7.jpeg
>
>


--------------------------------------------------------------------------------


> This attachment - 'DOTPLOT_unsorted.jpg' -  34.91 KBytes - can be viewed 
> at
> http://www.cyto.purdue.edu/MD-parts/62def7c7fe10f7697e00fc291358b32ab2249f9c.jpg
>
>


--------------------------------------------------------------------------------


> This attachment - 'DOTPLOT_59597.jpeg' -  24.67 KBytes - can be viewed at
> http://www.cyto.purdue.edu/MD-parts/d06c375adb89189a8fd2cbf9b0f34fd2bc908d64.jpeg
>
>


--------------------------------------------------------------------------------


> This attachment - 'DOTPLOT_59598.jpeg' -  23.17 KBytes - can be viewed at
> http://www.cyto.purdue.edu/MD-parts/43e39152176f8b49a2b4ee81816578e0218d7ef1.jpeg
>
>


--------------------------------------------------------------------------------


> This attachment - 'DOTPLOT_59599.jpeg' -  23.07 KBytes - can be viewed at
> http://www.cyto.purdue.edu/MD-parts/d2655060721347d778f9a78ee8cd420a6fd3e8f5.jpeg
>
>