Dear Eric and all colleagues,
Thank you for your replays to this question. I am pleased to hear that your
response corroborates what we at PharMingen Technical Service explain to our
customers regarding the issue of washing away free Annexin before analysis. We,
and others, have found it unecessary to do a wash step because unbound Annexin V
falls outside the scatter profile of cells.
Thank you very much to everybody who responded. We sometimes refer our customers
to the expertise of the members of this board if more information is needed.
Best Regards,
Dr. Marina Gumanovskaya
Technical Service Scientist
BD PharMingen
Eric Miller <millere@icrf.icnet.uk> on 04/12/2000 12:49:08 AM
To: cyto-inbox
cc: (bcc: PMGTechServices/SDCA/BDX)
Subject: Re: annexin V stain questions
There is one very good reason why the unbound stain causes no problems:
threshold. If you are thresholding on FSC then it is highly unlikely that
any unbound dye molecules will be large enough to trigger a pulse. Not
washing out unbound stain does feature in many protocols, such as
Vindelov's DNA protocol.
Eric P Miller
Edinburgh Medical Oncology Unit
"Everyone can be correct, all at the same time. That's the
thing about quantum."
Terry Pratchett, Lords and ladies
On Mon, 10 Apr 2000, Jamie Brewer wrote:
>
> I am writing to inquire about an annexin V stain I am used today from
Pharmingen. After
> calling the techinical support service as Pharmingen, I was given this E-mail
> address to correspond to with my unanswered question. The confusion comes in
that
> the Pharmingen protocol does not have me wash off any unbound annexin prior to
flow
> cytometry analysis. This is completely contradictory to any fluorescent stain
I
have ever
> used. Typically, the unbound stain fluoresces when analyzed, losing any
specificity the
> system had. My results seemed to turn out as expected today but I don't
understand why
> the unbound stain is not causing me a problem. I have been unable to find any
reference
> to the annexin being "active" only in the bound form as opposed to the unbound
form
> or any statement indicating the something in the binding buffer inactivates
unbound
> annexin. The technician at Pharmingen didn't have any idea why the unbound
stain is
> not causing a problem. Do you have any understanding of this? Also, the
protocol does
> not offer any option to fix the cells, something which I really need to do
since I
> am using potentially infectious human cells in a common flow cytometry
facility. The
> Pharmingen representative said that she knew of some people who had fixed the
cells
> and it worked alright but not as well as if the cells were not fixed. Do you
have
> any advice/suggestions?
>
> Please correspond to:
> Jamie Brewer
> jbrewer@hsc.wvu.edu
> Microbiology/Immunology Department, Mary Babb Randolph Cancer Center, West
Virginia
> University
>
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