Our laboratory has been using the standard acid-denaturation protocol for testing BrdU incorporation by flow, but we have been disappointed by the number of common epitopes destroyed during this process (e.g. CD23 on murine B-cells). We have been testing the DNAse protocol described by John Sprent's group, but with little success. All of the key reagents were recently purchased and work well in other assays. I am curious about two aspects of the protocol: 1) Does azide interfere with the DNAse enzyme activity? (we use azide in our common stock of 1% paraformaldehyde solution) 2) Does the source of DNAse make a difference? (we have been using molecular biology grade RNAse-free DNAse I) Any other suggestions or helpful comments on common problems would be greatly appreciated. We have already spoken with Dr. Sprent's lab and they have not had similar problems. -------- J. Paul Zimmer, Ph.D. Developmental and Clinical Immunology University of Alabama at Birmingham
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