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The following message was posted to: PharmPK
Dear all,
We are studying the disposition of a drug. The following organs or
tissues are obtained after injection:
liver, kidney, heart, spleen, lung, pancreas, brain, adrenal gland,
uterus, ovary, fat, musle, stomach, small and large intestine.
I tried to make homogenate for these samples but it's difficult to
some of them. Can I just take part of these tissues, weigh, cut into
pieces, solubilize overnight, treat with hydrogen peroxide before
LSC? I have no experience before so not sure whether it works well
or not.
I would appreciate greatly for any suggestion or idea on preparation
of these samples or other details on Liquid Scintillation Counting.
Ru Yan
OB/GYN
UTMB
email: ruyan.-at-.utmb.edu
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I would be careful and contact Amersham for methods/reagents
pertinent to tissue solubilization prior to LSC: they used to publish
excellent booklets on this topic. It would be advisable to study
these booklets (if they are still available) before starting the
work. You have to be careful and avoid producing effects like
chemiluminescence, which can interfere with the results.
Good luck!
Angus McLean
8125 Langport Terrace,
Gaithersburg,
MD 20877
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Hi Yan,
I have made homogenates for Brain,Heart,Liver,Lung,Spleen and Kidney
for LSC counting!
The procedure that I followed was to make a 10% homogenate in Tris-
HCl buffer
Liver, brain are fairly easy to homogenize therefore no need of
mincing those.
Spleen,heart, lung and kidney were minced and then homogenized in the
buffer.
Later, I added a tissue solubilizer to it to give me a clear solution
after digesting at 50 degrees. For colored samples, 100-200
microlitre of hydrogen peroxide was added to decolorize the samples.
This procedure proved very helpful to me and gave me reproducible
results!
Hope this helps!
Regards,
Kavita
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Dear Ru,
Most any LSC supply company has a ready-made cocktail for tissue
solubilization (e.g., Beckman, Amersham, Packard, and others). By the
way these firms are the very best source for practical information on
LSC methodology, including sample preparation, counting methods and
technology, etc.
In my experience, a digest of t-butyl ammonium hydroxide solubilizes
almost every tissue, including bone, teeth, muscle, and feces. This
works particularly well with samples placed in a warm water bath with
an orbital shaker for several hours. When solubilization is complete,
pipette the sample into a scintillation vial, add cocktail (Dimilume
from Packard has a chemiluminescence quench additive), add a couple
of drops of acetic acid to further reduce chemiluminescence. Dark
adapt overnight and count.
Best Regards,
David S. Farrier, Ph.D.
Summit Research Services
"PK Solutions" - for easy pharmacokinetics analysis
ADME/PK Software at www.SummitPK.com
68911 Open Field Dr.
Montrose, CO 81401
970-249-1389
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Yes solubilizer from Perkin Elmer also does a good job. And requires
heating to digest some tissues.
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Hi,
You will find below the procedure we apply in our lab. It's a part a
paper which we will published in the Journal of Pharmacy and
Pharmacology in few weeks :
"Plasma and tissues samples were treated with the same procedure.
100uL of plasma and carefully weighed samples (80-100ug for various
organs, 250 to 300ug for brain) were mixed 900 uL Soluene350(R)
(PERKIN ELMER, MA, USA) and incubated overnight at 60*C. Then,
samples were left one hour at room temperature for cooling, and 300uL
30% hydrogen peroxide were added to reduce quenching. A gentle
agitation allowed foaming reaction to subside during 30 minutes at
room temperature. Vials were finally incubated one hour at 60*C then
cooled to room temperature, added 10 mL of Hionic-Fluor (PERKIN
ELMER, MA, USA) scintillation cocktail. Radioactivity was counted
(during 20 minutes per sample) in a liquid scintillation counter
(Beckman, LS6000TA,Fullerton, CA)."
May this help,
Dr Makrem BEN REGUIGA, Paris
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