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While I do not have any direct experience with this, a user of my laboratory
used the BD CycleTest reagent kit on blood from desert tortoises (not lizards
but at least a reptile) and produced reasonable histograms with CV's in the
2-2.5% range on a FACScan. If you like to "home-brew" your reagents,
rather than buying a kit, a similar formulation to the contents of the
CycleTest reagents are given in Vindelov and Christensen, Cytometry 11(7):
753-770, 1990.
Another possiblity for stability problems could be related to the character-
istics of the instrument you're using. PI emission can be reduced by mixture
with the volume of sheath fluid that occupies the sample tubing when you
first start running your sample. As the sample runs, the sheath fluid is
gradually flushed out by the sample itself, and the PI concentration becomes
more constant. This can take up to 5 minutes or more on instruments with
long sample introduction tubes, like the FACS sorters. Possible solutions
to this problem are to (1) wait until stability is achieved, or (2) boost
the sample to flush out the line, or (3) change the sheath buffer. I
believe that the Vindelov & Christensen article mentions this, as well as
other hints at achieving stability in DNA analysis. Though the focus of
the article was on human samples, perhaps some of the material it discusses
might be useful.
-- Neal Benson | Phone: (904) 392-0008 Department of Pathology | FAX : (904) 392-4693 Box 100275 | Email: nbenson@nervm.nerdc.ufl.edu University of Florida | Gainesville, FL 32610 USA |
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