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@pharmingen.com [SMTP:pkodukula@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@pharmingen.com > > > > > "Barsky, Lora" <LBarsky@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@pharmingen.com [SMTP:pkodukula@pharmingen.com] > > Sent: Tuesday, September 21, 1999 9:18 AM > > To: Barsky, Lora > > Cc: jwaters@pharmingen.com; bobb@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@pharmingen.com > > > > > > > > > > "Barsky, Lora" <LBarsky@chla.usc.edu> on 09/07/99 11:40:54 PM > > > > To: Cytometry Mailing List <cytometry@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@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 > > > > > > > > >
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