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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