Cause of Sort 'impurity'

Moss, Delynn M. dmm3 at CDC.GOV
Tue Dec 4 12:09:53 EST 2001


Ray Hester wrote:

In trying to 'advertise' one area of our flow capabilities, I ran some of
the BD Calibrite red and green beads, sorted each population and then
re-analyzed the sorted populations. I intended to send these to the faculty
here to show how a typical 2-color cell sort might go.

As you can see in the first attachment, Fig. 1 is the unsorted sample, Fig.
2, the red bead sorted population upon re-analysis, and Fig. 3, the green
bead sorted population upon re-analysis.

In both of the sorted populations, the highest purity I can achieve is
around 96% because of some very small, non-fluorescent 'junk' that appears
in the lower left quadrant - representing about 4% of the events in both
sorted populations.

As you can see in the second attachment, there are some events by light
scatter that don't appear in the unsorted sample (Fig. 4), but do in the
sorted, re-analyzed samples (one is shown in Fig. 5).

At first, I thought this was from dirt/dust in the sort collection tubes -
I'm sorting directly into glass tubes without any medium or buffer because I
don't want to dilute the beads before re-analysis.  But I rinsed all of the
tubes before analysis and that didn't help.

I know this isn't that big a deal - unless you're trying to sell your
services - and then 4% 'contamination' might be important.

Has anyone else had this happen and, if so, what was the source of the
unwanted, very small, non-fluorescent particles?

Thanks for any help.






Ray,

You may have debris in the fluidics tubing.  Depending on what I am sorting,
it may take up to 20 minutes to clean out unsorted material in my tubing
using 0.2 micron filtered PBS, and still, I will have 10 - 20 events per
second being detected.  On occasion, I have cleaned the sample tubing with
10% bleach followed by PBS before checking my sorted material, which has
reached as high as 99%.  Dirty lines are likely your problem since events
are present in quadrant IV and I of Fig. 2 and Fig. 3, respectively, on your
sorted material.  Another possibility is to increase your FSC threshold if
you are triggering on FCS.  From Fig. 5, I see you can increase the FSC
threshold a few channels and eliminate some unwanted events before
interfering with the beads.  Lastly, do a drop-delay profile on your
calculated drop delay to make sure it is optimal.

Hope this is of some help.

Delynn Moss
CDC
Parasitic Diseases Division

 -----Original Message-----
From:	ray hester [mailto:rhester at jaguar1.usouthal.edu]
Sent:	Friday, November 30, 2001 5:37 PM
To:	Cytometry Mailing List
Subject:	Cause of Sort 'impurity'

 << File: SortFL.jpg >> << File: SortLS.jpg >> Hi,

In trying to 'advertise' one area of our flow capabilities, I ran some of
the BD Calibrite red and green beads, sorted each population and then
re-analyzed the sorted populations. I intended to send these to the faculty
here to show how a typical 2-color cell sort might go.

As you can see in the first attachment, Fig. 1 is the unsorted sample, Fig.
2, the red bead sorted population upon re-analysis, and Fig. 3, the green
bead sorted population upon re-analysis.

In both of the sorted populations, the highest purity I can achieve is
around 96% because of some very small, non-fluorescent 'junk' that appears
in the lower left quadrant - representing about 4% of the events in both
sorted populations.

As you can see in the second attachment, there are some events by light
scatter that don't appear in the unsorted sample (Fig. 4), but do in the
sorted, re-analyzed samples (one is shown in Fig. 5).

At first, I thought this was from dirt/dust in the sort collection tubes -
I'm sorting directly into glass tubes without any medium or buffer because I
don't want to dilute the beads before re-analysis.  But I rinsed all of the
tubes before analysis and that didn't help.

I know this isn't that big a deal - unless you're trying to sell your
services - and then 4% 'contamination' might be important.

Has anyone else had this happen and, if so, what was the source of the
unwanted, very small, non-fluorescent particles?

Thanks for any help.

Ray Hester
Univ. of South Alabama
rhester at jaguar1.usouthal.edu








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