Sort Questions

Mandy Boyd boydm at
Mon Jan 28 15:04:59 EST 2008

Hi Bryant, I run the same instrument as you. I find that resorted GFP
purity is not top notch because the cells are not so happy going through
the sorter twice and some die. I would bet that the GFP negatives are
really dead since most cells lose expression when they die. I add PI to
the reanalysis tube and gate on the live cells for best purity results.
Happy sorting!

Bryant Hanks wrote:

>Hi All
>I'm new to flow so I apologize in advance for what may be elementary
>We have a FACS Vantage SE running DiVa 4.0 with BD's Accudrop, ACDU,
and Pulse
>Plus options.
>After the deflection plates have warmed up an hour or so and the
fluidics have settled
>in, I optimize with accudrop beads and run a test sort.
>I run a FITC/PE calibrite bead mix, gate the separate populations and
do a 2 tube sort.
>The post sort purity check is excellent. FITC is 99+% and PE is 0% and
vice versa for
>each sorted tube.
>The problems occur with live cell sorts. An investigator will want to
sort the positive
>GFP (top) 25% and the negative GFP (bottom) 25%  of a cell population
into a 2 tube
>The post sort purity check can look pretty bad. While there may be a
>of the "sorted" population present, often the entire parent population
is present also.
>Sometimes this occurs with excessive, incurable "fanning" and
sometimes not.
>Any suggestions, explanations or ideas would be greatly appreciated.
Please feel free to
>point out the obvious.

Miranda Boyd
Flow Cytometry Operator
(503)220-8262 x56874
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