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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