Dear Jamie, as a clinician working along with a colleague with expertise in flow cytometry, and having had some experience in this field, I will make my humble contribution to this question! We work with sputum and bronchoalveolar lavage cells, staining with both annexin V (AV) and propidium iodide (PI). We had been concerned r.e. exactly the same issue, and particularly, were concerned that there was not an extra wash stage for the PI. My colleague with the previous flow cytometry experience would concur exactly with what you have said. In practice, we do wash after labelling. Anecdotally, I can say that we have not found much difference. We did run one experiment where we ran a sample where we had done an extra wash, and compared with those where we didn't wash. There seemed to be no difference in staining in this n=1 experiment. I seem to remember reading somewhere that washing labelling may affect the cell membrane permeability, so affecting PI uptake (and possibly also AV staining), tho' we haven't really seemed to see this in practice. I will see if I can find where I got this information, and see if I can firm up my 'vague recollection'! I know this doesn't really answer even the first half of your question, but I am interested to see that someone else has had the same thoughts on this. I would be very interested to hear what comes of any discussion, so that we can improve our practice too, if necessary. I am surprised that Pharmingen weren't able to offer help... Best wishes, Martin Kelly On Mon, 10 Apr 2000 15:29:22 -0400 Jamie Brewer <jbrewer@hsc.wvu.edu> 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 ---------------------- Martin Kelly Department of Clinical Biochemistry, Institute of Clinical Science, Grosvenor Road, BELFAST, BT12 6BJ. UK. Tel No: +44 (0) 28 9026 3267 Fax No: +44 (0) 28 9023 6543
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