Looks interesting, I'd be very interested to hear other peoples views. It looks like two problems, one with the secondary binding, and the other with the primary (iso control) binding to FcR. Having had blocking problems with FcR (especially FcRI) I took to using 5% AB sera as a blocking agent. Great providing that you are not using secondary's that recognise human IgG. My other cheat for human monocytes is to select antibodies that are mouse IgG1 wherever possible. Craig Turner. IGBRL ---------- From: simmmmer@yahoo.com To: cyto-inbox Subject: titration and blocking, a cry for help Date: Wednesday, March 22, 2000 22:29 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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