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
_________________________________________________________________
Get your FREE download of MSN Explorer at http://explorer.msn.com
This archive was generated by hypermail 2b29 : Sun Jan 05 2003 - 19:01:16 EST