BrdU with DNAse

Paul Zimmer (
Mon, 31 Mar 1997 12:03:04 -0600

Recently, I sent a message to this list asking about the Tough and
Sprent (1994) method for analyzing BrdU incorporation into lymphocytes
using DNAse. Several people wrote with inquiries about this method, so
I will pass on what I have learned. The complete method is published in
the Current Protocols in Immunology books (Unit 4.7). Like all
protocols, there is usually one step that must be carried out in a
special way to produce good results. In this case, the DNAse must be
purchased from Sigma as described in the protocol (we did not have luck
with a molecular biology grade, RNAse-free DNAse) and the frozen stock
of DNAse should be diluted in the recommended buffer IMMEDIATELY before
use (i.e LESS THAN 5 MINUTES). Our mistake appears to be that we were
preparing all the reagents before starting the protocol (including the
DNAse solution), which means the DNAse was sitting on ice for an hour or
more before being added to the cells. Other than that, follow the
protocol exactly and you should be able to get good results! Thanks
again for all the recommendations I received from the list.

J. Paul Zimmer, Ph.D.
Developmental and Clinical Immunology
University of Alabama at Birmingham Medical School

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