Hi Jamie, I certainly dont wash my samples after the annexin binding step - there may be an slightly increased background due to non-specific sticking of the annexin to negative cells but the true positives will still be obvious. It is best to avoid any unnecessary centrifugation steps as it may lead to differential cell loss of the apoptotic cells. Unbound annexin (or any antibody) will not cause a problem as it will be too small to trigger the machine. Regarding fixation, you presumably want to fix the cells prior to any staining? The trouble with this is you will lose the ability to separate the early apoptotic (FITC+/PI-) from the late (FITC+/PI+) ones. The time when the cells are passing through that early stage in an individual cell is quite short so it is better to avoid this! If you really have to fix, then you are limiting your choices to methods that can be performed on fixed cells eg subG1, TUNEL unless you use the 7AAD/AD technique to identify the dead cells prior to fixing and annexin staining. Derek 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 > ************************************************************************ Derek Davies Voice: (44) 0207 269 3394 FACS Laboratory, FAX: (44) 0207 269 3100 Imperial Cancer Research Fund, e_mail: derek.davies@icrf.icnet.uk London, UK mobile: 07790 604112 Web Page: http://www.icnet.uk/axp/facs/davies/index.html In tenebris lux *************************************************************************
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