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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