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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