Sort Questions

Julie Bertout julie.bertout at ibl.fr
Fri Jan 25 09:01:47 EST 2008


Hello,
I had the same problem.

When do you check the sort purity?
Because, in my hands, when I sort cells according to GFP, if I analyse 
them right away after the sort I have a bad purity (positive and 
negative cells) but when I analyse them after few days of culture the 
purity is very high. It is as if the GFP bleached during the sort and/or 
the cells need to recover... If your GFP expression can last few days 
after the sort, you can try to culture your sorted cells and analyse 
them. Of course it depends on the purpose of your sort but it can be a 
good control.

Concerning the nozzle, I always use low pressure and 100nm nozzle for 
cell line sort (FACSAria).

Please let me know if this helped.

Julie Bertout
Flow cytometry core
Institut Pasteur de Lille
France
+33 3 20 87 11 37


Bryant Hanks wrote:

>Hi All
>
>I'm new to flow so I apologize in advance for what may be elementary issues.
>
>We have a FACS Vantage SE running DiVa 4.0 with BD's Accudrop, ACDU, and Pulse
Processing
>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
sort.
>The post sort purity check can look pretty bad. While there may be a higher
concentration
>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.
>
>Thanks,
>
>Bryant
>
>
>
>  
>





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