Single cell sorting with the Aria

Good morning, 

We have been trying to sort single hematopoietic stem cells (very small
population - 1 cell / 100K cells) into both 72 well plates (10 ul/well)
and 96 well plates on the Aria without much success and was wondering if
anyone on the Purdue list serv has had any luck with sorting small
populations or single cell sorts on the Aria. Below is a short description
of our procedure & gating strategy:

We
> have been trying to sort murine HSC as lineage-/Sca-1+/c-kit+ cells on
>our new Aria without much success. We use a very bright GFP mouse (not the
>our new Aria without much success. We use a very bright GFP mouse (not the
>one from Jax) 
with 
>Sca-1-PE, PI, c-kit-APC and B220, Ter-119, CD3, Gr-1, Mac-1
>(all linked to biotin with SA-APC-Cy7). We also have an LSR-II on which
>we perform the
>same analysis
 without much trouble
>.

Our gating strategy is : 

1. n
>eutral density filter
s
> in 
both 
>FSC
 & SSC
2. threshold set on FSC and low (5000). Cell media has 10 mM Hepes and
0.5% FBS
3. 
>Gate FSC-Area vs SSC-Area on cells of interest
4. 
>Verify the Area Scaling Factor is "good" 
by adjusting PMT till we have a line of cells at a 45 degree angle on
SSC-Height vs SSC-Area
5. set first doublet discriminator by c
>reat
ing
> a child gate using SSC-H vs SSC-W
6. set second doublet discriminator by creating a third child gate using
FSC-H vs FSC-W
7. fourth child gate on PI- lin- cells
8. fifth child gate on Sca-1+ ckit+ cells
9. replot on FSC-A vs SSC-A to make sure they are cells of interest and a
tight population (which they are), then
9. sort fifth child population

we do 2 sorts: the first sort we do at a medium speed (aria sorting rate
=4) going about 20,000-30,000 cps. We then do a second sort on the first
sorted population at a lower speed ( aria sorting rate = 2 or 3) and more
dilute volume (~10,000 cells / ml). This lowers our abort rate to almost
zero. On the second sort, we sort single cell/well, then analysis the
second sorted population. 
in our single cell, 72 well plates (10ul/well volume), only 46% of wells
have a cell (N=5), the rest were empty. On the standard 96 well plates,
only 17% of wells had a single cell (N=4), the rest were empty (although I
could have missed some - finding a single cell in a 96 well is difficult,
but the results are still poor). 

On analysis of second sort, we find our purity varies from 46% to 96%. We
also have a number of events outside our FSC-A vs SSC-A population. These
could be cell fragments, but we add PI each time we sort and find we only
loss  2-5% of cells. They could also be microbubbles, but they are pretty
far out on the FSC-A vs SSC-A plot to be microbubbles. Our low plating
efficiency makes us believe that the Aria is sorting the cell fragments or
microbubles into well when it shouldn't be. On the advice of Mark Edinger
of BD, We have also tried to sort the fifth child population and inverted
gate population (ie a "NOT" gated population) to get eliminate the
"trash", but that hasn't worked either. Has anyone had in luck with
sorting with invert gates on the Aria? 

Thanks
David

************************************************
David Holtzclaw, Ph.D.
Emory University Pediatric Hematology-Oncology-BMT
Dental Building, Room 440
1462 Clifton Rd. 
Atlanta, GA 30322
Voice: 404.727.1422
Fax: 404.727.4859
Email: jholtzc@emory.edu
Website: http://userwww.service.emory.edu/~jholtzc/
***********************************************

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