I think perhaps Marv's request for help is lost in the thread. However, I
could be wrong...
It sounds like to me the sheath fluid, if matched to the cell of interest
(in this case, fresh water)
one runs into the problem of the stream not charging adequately for
sorting.
Pure deionized water does not charge to suit our sorting needs (as can be
theoretically deduced and pragmatically tested when cleaning and rinsing
with sheath-ddH20 and running a test sort),
thus a sort decision does not happen as we would like. I have never
sorted such a sample requiring fresh water. However,
I would suggest Marv try mimicking fresh water as much as possible, while
thinking about compounds found in fresh water that would hold a charge.
Some elements and compounds that come to mind are low concentrations of
calcium, magnesium, and sodium. Alternatively, in nature, fresh water
contains tannins and phenols derived from plant decomposition. As well,
it is likely many free amino acids are dissolved in fresh water, and would
likely
hold a charge. Perhaps a sterile solution capable of holding a charge
could be derived from using one or more of the above without stressing the
protozoan (thus preventing the release of GFP).
My Regards,
Christopher Boyce
Development Scientest
BCI Immunomics
JTrotter@PharM
ingen.com To: "Cytometry Mailing List"
<cytometry@flowcyt.cyto.purdue.edu>
cc:
12/21/01 02:06 Subject: RE: [GFP Retention]
PM
It is always advisable to match the sheath and sample as closely as
possible to minimize any differences in refractive index. Otherwise,
scatter information may suffer significantly, depending on the differences
in refractive indices. Sorting fresh water protozoans in a saline sheath
will result in a near sheath salinity after deflection (and mixing of
sample and sheath). Probably not a good idea... No point in arguing about
diffusion during the transition through the laser beam(s) if there is
mixing immediately afterwards.
Joe
Andrew Beernink <ABeernink@novasite.com> on 12/20/2001 02:49:45 PM
To: cyto-inbox
cc:
Subject: RE: [GFP Retention]
If the flow is laminar, and we assume that diffusion of salts from sheath
to cell suspension medium (water) is negligible (bad assumption?), the
sort decision is made while the protozoan is still in fresh water, so it
shouldn't change our ability to sort, only to reanalyze our sorted sample,
assuming our (bad?) assumption about diffusion is correct.
Ideas? Feedback? (please, no gunfire!)
Andrew
Andrew Beernink
Research Scientist - Flow Cytometry
Novasite Pharmaceuticals, Inc.
11095 Flintkote
San Diego, CA 92121
(858) 638-8584
(858) 597-4943 fax
-----Original Message-----
From: James Marvin [mailto:jmarvin@flowcity.bsd.uchicago.edu]
Sent: Wednesday, December 19, 2001 12:17 PM
To: cyto-inbox
Subject: [GFP Retention]
Hello,
We have a client who is trying to sort a fresh water protozoan that
expresses GFP. They are very worried about stressing the cells. They have
found that stressing these cells causes them to release their GFP and thus
show up negative. I was wondering if anybody has determined the minimum
concentration of salt that can be used in the sheath fluid that will still
hold a charge. Presently we use a 1X PBS solution consisting of NaCl, KCl,
Na2HPO4 7H2O, and KH2PO4
Thanx in advance, Marv
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