Re: Sorting GFP

From: Mark A. KuKuruga (kukuru@umich.edu)
Date: Fri Mar 26 1999 - 09:53:51 EST


Ann Williams wrote:

> When sorting cells for surface markers we get 99+% purity.  When we sort GFP+  from
> trypsinized cultures the percentage is less.  We do get better results with GFP+  from
> suspension cultures.
> We would like to know if others see this with GFP.  Any suggestions on how to increase
> the purity?
>

Ann,We've had similar experiences with adherent / stickier cell types.  You said it
yourself . . . you do better with suspension cultures because these cell types naturally
exist as single cell suspensions.  The trick is to get your adherent cell types to act the
same . . . not an easy task.  So, the problem comes when you sort a positive, but that
positive is connected to a negative.  This "aggregate" then dissociates after sorting, and
you get a "negative" contamination.
One solution to this is to improved aggregate correction.  The Elite ESP system has a Time
of Flight function (duration of event in beam) that, when measured against a peak signal
(usually forward light scatter) allows for aggregate selection.  We have validated this
method by sorting the "singlets" and "aggregates," then evaluating by microscopy.  This
method has proven very accurate.  Example; lung homogenates from mice then surface stained
were notorious for "negative" contamination upon sorting.  We would typically double-sort
these samples, and at best achieve about 90-93% purity, from a basis of about 5-10% . . .
not too good.  This was also problematic since 1) the viability was adversely effected,
and 2) cell loss was doubled.
Including Time of Flight in our sort equation has allowed us to sort to 95-99% purity in
one pass, with comparable sort efficiency.
Now, you didn't mention your instrument, so I don't know if this is an option for you, but
thinking about you source of contamination in this fashion may give you some help.
In any event, be sure to check your samples for aggregation if you're having problems.  If
you do see aggregates, modifications to your tissue handling / suspension buffers may
eliminate this.
Good luck.
MAK.

> Ann Williams
> NHLBI, NIH
> williama@gwgate.nhlbi.nih.gov



--
Mark A. KuKuruga, Managing Director
University of Michigan Core Flow Cytometry
kukuruga@medmail.med.umich.edu



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