------- Forwarded message
Posted: Wed, 03 Dec 97 05:00:01 -0500
Date: Wed, 03 Dec 97 18:37:01 -0500
Author: sam~witherspoon
Subject: help with isolating nuclei from mouse intestinal tissue for analysis
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Greetings Flow Folks,
Karren (and I) would appreciate input from you DNA guru's out there.
Please check the protocol and send your comments to Karen,
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Hello all-
I am trying to optimize a protocol for isolating nuclei from 3 cm sections
of mouse small intestine. These nuclei are to be run over a FACSort for
analysis of PI (propidium iodide) and BrdU (bromo-deoxyuridine)
incorporation into DNA. My current procedure yields nuclei preps which
significantly vary from experiment to experiment in the PI profile that I
see: Rather than seeing a PI profile with 2 peaks representing the G1 and
G2 phases of the cell cycle (which I set at 200 and 400 on the X axis), I
am seeing a series of peaks which lie at 200, 400, 600, 800, and 1000...
.continuing off-scale. The percentage of the events collected that lie
within the first two peaks (presumably G1 and G2) varies from between 20% to
75% of total events from one experiment to another (though it is consistent
from one sample to another within a given experiment). I have not been
able to identify changes in experimental handling of samples that correlate
with the PI profiles that I see. Gating for singlate events on a FL2-W
vs FL2-A plot does not change the PI profile. As the tissue is normal
intestinal tissue, there is no reason to assume that the nuclei would
contain any amount of DNA other than the usual 2N-4N associated with a
normal cell cycle.
The protocol that I have been using is as follows- The animals are dosed
with BrdU (120 mg/kg) i.p. one hour before sacrifice. At sacrifice,
approx 3 cm of intestine is excised and put into cold 70% ethanol. The
tissues are stored for random periods of time at -20C in the ethanol. The
tissues (still in ethanol) are disrupted using a Polytron homogenizer at
maximal speed for 30 sec. After centrifugation of an aliquot of the cell
suspension, the cell pellets are suspended in 2.5% pepsin in 0.1 N HCl
and incubated for 45 min at 37C with mixing every 10 min to release the
nuclei. After centrifugation, the nuclei pellet is suspended in 2 N HCl
(to hydrolyse the DNA into single strands) and incubated at 37C for 20 min.
Sodium borate is added to neutralize the acid and the nuclei are pelleted
again. Following a wash with IFA buffer (10 mM Hepes, 150 mM NaCl, 4%
fetal calf serum, 0.1% sodium azide) containing 0.5% Tween 20, the
nuclei are incubated with Anti-BrdU antibody (at 1:5 in IFA) for 30 min at
room temperature in the dark. Following washing with IFA/Tween, the
nuclei are incubated with 100 ug/ml RNAse A in IFA for 15 min at RT in the
dark. Then PI is added to a final concentration of 10 ug/ml. The nuclei
are incubated for at least 15 min at RT in the dark before running over the
FACSort.
Microscopic examination of the nuclei reveals a suspension of nuclei that
can range from mostly single nuclei to mostly clumped nuclei, though I've
yet to identify all the possible manipulations of the sample that encourage
the clumping. The nuclei always clumped in PBS. I no longer use it.
The centrifugation speed is critical. Too hard and the nuclei cannot be
resuspended as single nuclei. Sometimes there is a lot of debris (or
something!!) in the samples OR they are very sticky and they clog the FACSort
SIP tube. Running the preps thru 40 uM filters before running over the
FACSort usually eliminates the clogging problems, though it does not
change the PI profile at all.
I've had great experience with this protocol when I use it on fixed single
cell suspensions from tissue culture cells. So, I suspect the
difficulties are arising in the tissue preparation/disruption end of things,
though I would be grateful for comments on any part of the procedure. Can
any of you point out the problems in this protocol, recommend changes that
would improve the reproducibilty of the preps, or otherwise help me out
with this? Please keep in mind that the intent of these experiments is to
run multiple groups of treated animals at once - so minimal experimental
size would be 30 samples or so. If possible, it would be nice to have a
protocol that the sample manipulations are do-able within a standard workday
on such a number of samples.
Thanks for any help you can give.
Karen Dold
kd15397@glaxowellcome.com
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