Hi everybody, We're doing Annexin staining on subsets of lymph node cells obtained after collagenase/edta digest. We use Annexin V-PE or anti-bcl-2-PE(Pharmingen) in combination with 2 MoAb markers (in FITC and RPECy5). I have several nagging questions: 1. This may sound quite trivial, but was is the influence of SODIUM AZIDE (as in your average FACS washing/staining buffer) on the final Annexin staining? I could leave out the azide in the washing buffer, but it's still present in minute quantities in the MoAb dilutions used prior to the Annexin... any effect on cell apoptosis? 2. During staining of such cell suspensions with the MoAbs, we preferably use a staining buffer containing 5mM EDTA (to avoid lympho-APC reaggregation). Of course I use the Ca-rich Annexin binding buffer for the final step, but does the previous presence of EDTA have any lingering effect on the quality of the Annexin staining? 3. How critical is temperature for the Annexin staining? 4. Same thing for bcl-2 staining: how is it influenced by azide? 5. Finally, is PFA fixation beneficial or detrimental to the quality of Annexin V staining? Any answers or comments will be greatly appreciated and will help others as well I hope! Karim Y. Vermaelen Resp. Diseases, Pulmonary Immunology Ghent University Hospital, Belgium
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