Hi Jeff,
As far as I understood, both methods induce DNA damage by photolysis. The
SBIP approach, however, depends on the TdT enzyme to make use of it, whereas
the UVID method puts the cells under a hypotonic stress to detect the
remaining BrdU with an antibody. Photolysis by itself doesn't seem to be
capable to allow the detection of BrdU.
Best regards,
Michael
>From: "Harvey, Jeff" <jharvey@guavatechnologies.com>
>To: Cytometry Mailing List <cytometry@flowcyt.cyto.purdue.edu>
>Subject: RE: Help about BrdU
>Date: Wed, 25 Apr 2001 14:52:22 -0700
>
>Hi all,
>
>SBIP does not, in and of itself, require the use of TdT. In fact, SBIP and
>UVID are identical in their use UV light to induce DNA strand breaks at the
>sites of BrdU incorporation. The UVID paper cited below describes the
>subsequent direct labeling of these BrdU moieties with a FITC-conjugated
>anti-BrdU antibody. The use of TdT to add additional BrdUTP moieties to
>the
>strand break sites is aimed at substantially amplifying the signal
>obtained,
>by allowing the attachment of many fluorochrome-conjugated anti-BrdU
>antibodies to the multiple BrdUTP's at these sites. This improves the
>sensitivity of detection of the S-phase cell population.
>
>Best Regards,
>
>Jeff Harvey
>Guava Technologies, Inc.
>
>-----Original Message-----
>From: Gretchen Lawler [mailto:GRETCHEN@flowcyt.cyto.purdue.edu]
>Sent: Wednesday, April 25, 2001 6:22 AM
>To: cyto-inbox
>Subject: RE: Help about BrdU
>
>
>
>Current Protocols in Cytometry has just published a unit on UVID by
>Dr. Hammers in Supplement 16.
>
> >
> >
> > There is an even more advanced technique now than the SBIP approach. It
>uses
> > no enzymes at all. No DNAse or TdT (SBIP). Instead, it only employs a
>short
> > irradiation period with UV-B and then subjects the cells to a hypotonic
> > buffer. It's called UVID (Ultraviolet-Induced Detection). I saw some
>people
> > in Texas using it - it works great !!
> >
> > Here is the paper:
> >
> > Hammers HJ, Kirchner H, Schlenke P. Ultraviolet-induced detection of
> > halogenated pyrimidines: simultaneous analysis of DNA replication and
> > cellular markers. Cytometry 2000 Aug 1;40(4):327-35
> >
> > Best regards,
> >
> > Michael
> >
> >
> >
> > >From: "Harvey, Jeff" <jharvey@guavatechnologies.com>
> > >To: Cytometry Mailing List <cytometry@flowcyt.cyto.purdue.edu>
> > >Subject: RE: Help about BrdU
> > >Date: Wed, 18 Apr 2001 12:39:12 -0700
> > >
> > >One additional note:
> > >
> > >You may also want to consider the BrdU kit from Phoenix Flow
> > >("Absolute-S").
> > >This one also gives very good results and has the unique attribute of
>not
> > >requiring DNase, HCL or heat treatment to make the incorporated BrdU
> > >accessible for labeling. Instead, a technique developed in Dr.
> > >Darzynkiewicz's lab, strand breaks induced by photolysis (SBIP), is
>used
>to
> > >accomplish this. There is a description of the technique in Howard
> > >Shapiro's Third Edition of Practical Flow Cytometry (p325).
> > >
> > >Regards,
> > >
> > >Jeff Harvey
> > >Guava Technologies, Inc.
> > >
> > >
> > >
> > >-----Original Message-----
> > >From: Carmen Raventos-Suarez [mailto:carmen.raventossuarez@bms.com]
> > >Sent: Tuesday, April 17, 2001 1:09 PM
> > >To: cyto-inbox
> > >Subject: Re: Help about BrdU
> > >
> > >
> > >Dear Martha:
> > >Just a note to support the use of the BrdU Pharmingen kit that utilize
> > >DNAse instead of the HCl as a good method for BrdU incorporation. It
>has
> > >also the advantage that using 7-AAD can allow a third parameter
>(surface
>or
> > >internal marker) to be evaluated.
> > >In our hands it gives excellent results, 30 min pulse for cells around
>the
> > >24h cell cycle is good, longer pulse times for slow growing cells. Our
> > >resolution for DNA phases is also good even when a third color is used.
> > >Carmen
> > >
> > >Martha Mesa - Microbiology wrote:
> > >
> > > > Dear sir,
> > > >
> > > > In my laboratory, I am studying the effect of some compounds on
>tumoral
> > > > cell cycle
> > > > by PI staining. In order to know if the antitumoral effect observed
>is
> > > > due to a
> > > > decrease in DNA synthesis I am trying to set the BrdU incorporation
> > > > assay, by I
> > > > have several questions; some of them are:
> > > >
> > > > 1. For cells with doubling time of 22 (k562) and 72 hours (KG1a),
>how
> > > > long must
> > > > be the pulse? I have used BrdU 10 uM for 8 hours; I have only
>obtained
> > > > low
> > > > intensity in fluorescence some times.
> > > >
> > > > 2.Some protocols recommend more HCl. I have used 2M, 3M and 4M. With
>4M
> > > > the BrdU signal improves but phase resolution with PI disappear. I
> > > > would like to
> > > > know the effect in each phase; how can I obtained that?
> > > >
> > > > Thanks in advance,
> > > >
> > > > Marta Mesa
> >
> > _________________________________________________________________
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> >
>
>
>
>Gretchen Lawler
>Purdue Cytometry Laboratories
>(765) 494-0757; fax (765) 494-0517
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