Dear all: Someone came to use my Facscalibur with annexin2 staining on mononuclear human cells. I acquired and analyzed them. Problems: 1. the person has no experience in staining with 2 step reagents. 2. the controls are absent in this run due to #1 3. the analysis is hindered by #1, #2 overview - the person separates monocytes with magnetic beads, checked for purity with CD14, that part works. Those are then stained with a monoclonal rabbit anti human Annexin2, followed by goat anti rabbit FITC. There is highly positive FITC signal in all tubes. I recommended that she runs an isotype control - rabbit anti rat igg, followed with goat anti rabbit fitc. The resutls are posted, as histograms: http://www.cd4cd8.com/titration1.html (isotype control) Note that none of the cells are in M1.. I could fudge with voltage and such but the instrument was JUST calibrated prior to this acquisition. http://www.cd4cd8.com/titration2.html (annexin2/FITC) Note the FITC GAR alone - that is definitely a start to this riddle. Questions: 1. what is a good control cell line for annexin2 expression? 2. ANY recommendations for general 2 step staining - my hunch is that her blocking is too weak.. also any help regarding titrations would be appreciated... Finally if someone feels my acquisition could be augmented to make this work - voltage settings, compensation, etc.. Thank you very much for your time. Best Regards, Maciej PS I have not updated my site, especially the cytokine cheat sheet, but I will right after MCAT :) __________________________________________________ Do You Yahoo!? Talk to your friends online with Yahoo! Messenger. http://im.yahoo.com
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