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Dear Paul,
You might want to try a protocol described in Carayon and Boyd (J. Immuno.
Meth. 147, 225-230, 1992), which uses a gentle fixation with
paraformaldehyde and permeablization with Tween 20 followed by DNase
treatment. The method has worked very well in our hands for
phenotype-specific measurement of BrdU incorporation in mouse T
splenocytes labeled for CD3, CD4, etc. No heating or acid-denaturation is
used, which preserves surface marker expression and prevents the
resulting cell damage and descrease in yield sometimes seen in some cell
types. We immunophenotype prior to paraformaldehyde fixation and
permeablization.
Bill Telford
Department of Pathology
University of Michigan Medical School
On Thu, 27 Feb 1997, Paul Zimmer wrote:
>
> 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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CD-ROM Vol 3 was produced by Monica M. Shively and other staff at the
Purdue University Cytometry Laboratories and distributed free of charge
as an educational service to the cytometry community.
If you have any comments please direct them to
Dr. J. Paul Robinson, Professor & Director,
PUCL, Purdue University, West Lafayette, IN 47907.
Phone: (765)-494-0757;
FAX(765) 494-0517;
Web