Doug,
I am not sure what the "accepted" standard is, but I'll offer the following thoughts based on our experiences.
Using beads as surrogates to test the efficiency and prescision of the sorter configuation makes sense
to me provided:
1 - the ratio of particle size to stream size is similar - the prescision of droplet placement running hematopoietic
cells will be different than when 2 micon beads are run, for example.
2 - the sorter is *very well tuned* in terms of phasing and drop delay - small errors here can impact significantly whether the sorting electronics judge correctly which drop an event will partition into. The first symptom of small
errors in delay time is reduced purity and yield. BDIS sorters, for example, use Counter mode to sort into
96 well plates(ACDU/CloneCyte). This adds another 0.5 drops to the apparent drop deflection packet to
"ensure" counting accuracy. So, using Counter mode to establish the drop delay profile adds 0.5 drops of
imprecision that needs to taken into account timing the system (we time our BDIS systems in Normal-R, then
change to Counter for 96 well sorting - and we usually run at slow to moderate speeds).
3 - the frequency of sample "doublets" is taken into account. We've all had experiences with sorting a small % of
positive cells when an occasional negative cell will be stuck to a (+) and be sorted as a single (+) cell. Sorting
the biological sample onto slides and looking with a fluorescent scope has clarified some questions when
sorting for negatives one side and positives the other side gave different purities upon reanalysis. Sample prep
seems the biggest factor here in getting "single cells" - concentrated samples are usually easier for us to
get good results with (smaller sample core diameter gives better "doublet" discrimination). The most difficult
for us are samples where the subset is small (< 1%) and tends to have "sticky" negatives. This is when we
focus more towards the sample buffer. The sorter coincidence circuits usually do a good job with separate
particles that appear in the beam together, but often fail to recognize two small cells "stuck" together
as a doublet. This is often why a (-) sort will be 99.5% pure and the (+) 97% pure sorting left and right
with identical timing with a 50/50 ratio of (-) to (+) in the sample. Two (+)'s are twice as bright, two (-)'s
sort as (-), but the (-)/(+) gets sorted as a single positive. In 96 well single cell sorting, any
sticky doublet is a problem, so looking under a scope at what you are selecting is essential (you would
miss the double negative with a reanalysis by flow alone, for example).
In summary, we do use beads to check the prescision of the sorters. They are usually *very* good at putting 10
micron beads where we tell them to. We monitor the sorting efficiency of the biological samples as well to check
purity, frequency of doublets, etc., and rely on that to tell what we need to do to try and get samples as good
as the beads. This often is only a matter of adding DNAse to the sample buffer, using a bigger nozzle for
drop stability, etc..... If you're concerned about "doublet" errors, you'll need to do the obvious and sort
quite a few into a tube for reanalysis using the same sort mode and deflection envelope as when placing
them into the 96 well trays. We also do "test" runs on top of the 96 well plates with the lids on and examine
those lids under a scope for doublets and placement. Noise in the sorted droplet placement is an indicator
of the sample prep causing instability. This is often remedied with a larger nozzle (we use 70, 80, 90
and 100 micron nozzles) and attention to the sample buffer and preparation. I'd like to know what you end
up doing to control for all the issues you must be facing.
We often sort a scatter distribution we suspect will contain doublets onto a slide to ensure we're doing the "right"
thing when setting up prescise sort regions. Not an easy issue if an error occurs 2% of the time, and 2 wells on
your 96 well plate do something "interesting". This is where one might need to sort 1000's of cells into a tube for
reanalysis to guage the frequency of an error to expect on the 96 well plates.
Best Regards,
Joe
----- Original Message -----
From: Doug Dooley
To: Cytometry Mailing List
Sent: Thursday, July 01, 1999 4:42 PM
Subject: single cell sorts
Fellow flowers:
What is the accepted standard for judging the success of "single cell" sorting into 96 well trays? That is, how does one verify that >95% of the wells received only one cell. We are sorting hematopoietic cells on to stroma. Therefore, I do not feel that we will be able to see the sorted cells afterwards. In fact, finding single cells in 96 well trays lacking stroma can be difficult. Is it "kosher" to use fluorescent beads as surrogates? Or, are beads not good representatives of the ability of the sorter to pick out single cells for deposition in a well. Your critical thoughts will be appreciated. Thanks.
Doug Dooley
Stem Cell Lab
American Red Cross, Portland, OR
dooleyd@pdxredx.org
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