We always wash our cells after incubation with Annexin V. I suspect that you will get a better signal to noise response (better separation between positive and negative) if you wash the cells. It may, however, not be as significant as one might think. A light fixation with paraformaldehyde will render the cells cooked with membranes intact. I would suggest a starting point of 1% for 5 minutes. I will assume you are already using PI to gate out (count) necrotic cells, which will have leaky membranes. If you see this number as being higher than an unfixed control then you will have to adjust your fixation procedure accordingly. Remember, you can also keep increasing the percentage of paraformaldehyde and use the above procedure to assess if you are affecting the membranes (increasing PI+). The higher the paraformaldehyde the greater your peace of mind. Hy humble opinion. At 03:29 PM 2000-04-10 -0400, 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 > > - Derek Schulze Flow Cytometry and Confocal Microscopy Core Facility Cancer Research Labs Queen's University Kingston, Ontario Canada
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