FW: caspase-3

Barsky, Lora LBarsky at chla.usc.edu
Wed Oct 6 17:02:05 EST 1999


For those of you who were following the Caspase-3 thread and those of you
who contacted me directly, here's the information regarding Caspase-3 and
Annexin V that I received from Pharmingen.  Any furthur questions, please
contact Padma at Pharmingen. Padma was most helpful.
Lora

> -----Original Message-----
> From:	pkodukula at pharmingen.com [SMTP:pkodukula at pharmingen.com]
> Sent:	Monday, October 04, 1999 10:14 AM
> To:	Barsky, Lora
> Subject:	RE: caspase-3
> 
> 
> 
> Dear  Lora,
> 
> Thank you for your interest in PharMingen's products.  This is in reply to
> your
> question about the caspase 3 and the annexin V staining.  We are releasing
> a kit
> for the BrDU and cytokine staining.  But the annexin V is also known to
> work
> with the caspase 3.  This is not something that we routinely quality
> control in
> our company but we have had input from customers who have used this
> method. The
> annexin V binding to phosphatidyl serine is dependent on the CA++ ion
> concentration and so a Ca++ containing buffer is used for the staining of
> annexin V.  After the annexin V staining the cells are washed twice in
> Ca++
> containing buffer can be fixed in 4% paraformaldehyde for 20 min at RT and
> then
> wash and  immediately stain intra-cellularly for caspase 3.
> Again this is not a protocol that we routinely quality control and you
> make have
> to troubleshoot it.
> Please let us know if you have any additional questions.
> 
> Best regards,
> 
> Padma Kodukula  Ph.D.
> Technical Service Scientist
> Ph. #  800-825-5832 X 4473
> E-mail  pkodukula at pharmingen.com
> 
> 
> 
> 
> "Barsky, Lora" <LBarsky at chla.usc.edu> on 09/23/99 12:22:05 AM
> 
> To:   Padma Kodukula/SDCA/BDX
> cc:
> Subject:  RE: caspase-3
> 
> 
> 
> 
> Hello,
> 
>      Thanks for reading the cytometry discussion list.  I appreciate your
> information regarding cytokines and BRDU but, my comment was to answer a
> question of Caspase-3 staining, more specifically Caspase-3 and annexin V
> staining of the same sample.  Now I don't get many opportunities to set up
> stains, I pretty much run the sorter and trouble shoot people's
> experiments.
> Umm, what I would like to know, while I have your interest is the
> following:
> 
>      I spoke with David Sehy, when he visited USC for a
> cytokine/apoptosis workshop.  He said that the kit for Caspace 3 and
> Annexin
> V from Pharmingen could be used together to double stain cells but you
> need
> one of them to be done in the presence of Ca++(?).  I can't find my notes
> anywhere.  If you could confirm this one way or another, it would be a
> great
> help.  Many people from the list have contacted me about this and I didn't
> have the time to investigate further.  I would be happy to post the
> information on the list to your credit.
> 
>      Thank you,
> 
>      Lora Barsky
> 
> > -----Original Message-----
> > From:   pkodukula at pharmingen.com [SMTP:pkodukula at pharmingen.com]
> > Sent:   Tuesday, September 21, 1999 9:18 AM
> > To:     Barsky, Lora
> > Cc:     jwaters at pharmingen.com; bobb at pharmingen.com
> > Subject:     RE: caspase-3
> >
> >
> >
> > Dear  Lora,
> >
> > I am responding to the request that you had regarding the staining of
> > intra-cellular cytokines together with a apoptosis marker such as BrDU.
> > We are
> > developing the BrDU kit which will allow for staining for a cell surface
> > marker,
> > intra-cellular cutokine and the BrDU all at the same time.  This BrDU
> kit
> > will
> > be available from PharMingen in the next month and it will come with a
> > detailed
> > protocol and the reagents required to do the assay.
> >
> > Please let us know if you have any additional questions.
> >
> > Best regards,
> >
> > Padma Kodukula  Ph.D.
> > Technical Service Scientist
> > Ph. #  800-825-5832 X 4473
> > E-mail  pkodukula at pharmingen.com
> >
> >
> >
> >
> > "Barsky, Lora" <LBarsky at chla.usc.edu> on 09/07/99 11:40:54 PM
> >
> > To:   Cytometry Mailing List <cytometry at flowcyt.cyto.purdue.edu>
> > cc:    (bcc: Padma Kodukula/SDCA/BDX)
> > Subject:  RE: caspase-3
> >
> >
> >
> >
> >
> >
> > Hello all,
> >      I recently attended a apoptosis/cytokine workshop sponsored by
> > Pharmingen in July.  One of the technical application specialists said
> > they
> > (Pharmingen) were gathering data to support that it was possible to run
> > both
> > tests on the same sample,  one followed by the other, provided you set
> up
> > the samples a particular way.  Maybe a call to customer service, or
> > technical application support, would help.  Of course, this data was not
> > published yet.
> >
> >      Good Luck
> >
> >      Lora Barsky
> >      Children's Hospital Los Angeles
> >
> > > -----Original Message-----
> > > From:   Maryalice Stetler-Stevenson [SMTP:stetler at box-s.nih.gov]
> > > Sent:   Tuesday, September 07, 1999 6:35 AM
> > > To:     Cytometry Mailing List
> > > Subject:     Re: caspase-3
> > >
> > >
> > > We also have tried this antibody in parallel with Annexin V without
> > > success
> > > but haven't done a time course, etc to determine if there are
> biological
> > > reasons this isn't working for us. I would be interested in others
> > > experience with this antibody and any tricks people may have used to
> get
> > > it
> > > to work better.
> > >
> > >    Maryalice
> > >
> > > >Dear all
> > > >
> > > >We have been using a anti-Caspase-3 rabbit polyclonal ab conjugated
> > > >with PE (this is supposed to bind to the active form only) by flow.
> > > >We've looked for caspase-3 activation in camptothecin or etoposide
> > > >treated Jurkatt cells (cytospins confirm apoptosis in these
> > > >cells) after fixation and perm, without success. This ab is from
> > > >Pharmingen.
> > > >
> > > >Does anyone have experience with looking for caspase-3 activation by
> > > >flow ? Are there other antibodies available ?
> > > >
> > > >Any comments would be much appreciated.
> > > >
> > > >Fran Gibson
> > > >Dept Haematology
> > > >St. George's Hospital Medical School
> > > >London
> > >
> > > Maryalice Stetler-Stevenson
> > > Director Flow Cytometry Unit
> > > Laboratory of Pathology, NCI, NIH
> >
> >
> >
> 
> 
> 



More information about the Cytometry mailing list