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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