Hi Karim, funny you should ask about these issues, as I was wondering myself just the other week. I have done one experiment to test the effect of azide (0.04% in wash buffers) on annexin staining in lymphocyte populations, and it seems that the presence of azide slightly reduces the number of annexin-positive cells (27% reduced to 19%). However, as I said, this was only one sample, so I wouldn't put too much weight on it. (although I do have a reference showing that azide inhibits apoptosis in Jurkat cells, so there may be something to this...?) With regard to fixation in PFA, I have found that dissolving the PFA (1%) in calcium-containing annexin binding buffer gives good fixation (scatter profiles are as expected), and I see approximately what I would expect in terms of annexin staining, although I am yet to compare fixed cells with un-fixed cells. However, when I used 1%PFA in PBS, the scatter profile was a mess, and I couldn't gate my lymphocyte population. I think the calcium buffer reacted somehow with the PFA/PBS, as there was a white precipitate in my tubes. Sorry it's all a bit vague, but I hope this helped in some sort of way... Lisa Karim Vermaelen wrote: > > 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 -- Lisa M Gale Department of Microbiology & Immunology University of Adelaide ph +61 8 8303 4630 fax +61 8 8303 4362 email lisa.gale@adelaide.edu.au
This archive was generated by hypermail 2b29 : Sat Mar 10 2001 - 19:31:26 EST