Hi Jos, there are number of CD34 antibody conjugates available from which to choose. The class III clones like 581 and 8G12 work very well in FITC, PE, APC and (some) PECy5 forms. However, I have tested a couple of PECy5 conjugates of other clones over the years that were not well manufactured and these reagents (the PE was probably over-conjugated with Cy5 moieties) stuck to everything with an Fc receptor. The 581 PECy5 conjugate from Beckman Coulter is very good however and seems reliable lot to lot. I would not use indirect fluorescence for CD133. Some advice offered over the last few days may make life more tricky than it needs to be. For example if you use unlabelled CD133 followed by a labelled secondary 'anti-mouse' conjugate, you then MUST wash unbound secondary reagent away and then BLOCK any unoccupied mouse binding sites with normal Mouse Ig before introducing more directly conjugated murine antibodies. Failure to do this will result in all sorts of staining artefacts with virtually all CD133+ cells staining with all other conjugates in your cocktail. One other thing; titrate everything before you finalise your cocktail, as well as use robust Fluorecence Minus One controls. You could check out Current Protocols in Cytometry Unit 6.4 to see how we performed simpler versions of what you are about to try to set up. I would also suggest the use of CD45/CD34 and ISHAGE protocol gating strategies to identify bona fide CD34+ cells in the first place. best, Rob Sutherland University Health Network Toronto On 20-Dec-07, at 10:10 AM, Jos Bosch wrote: > Dear Flowers, > > > > Am setting up a panel on a 6-color FACScanto to characterize stem > cells in peripheral blood, and running into the usual planning > problem on what fluorochromes to assign to particular antibodies. > The main hassle is caused by the fact that CD133 (miltenyi) and KDR > (R&D) are only available in PE and APC, so have to work around these. > > > > To make a long story short, there are two options I seem to be left > with: > > 1) Substitute CD34 PE (clone 8G12, BD) for PerCP. > > Arseniev (1999) concluded PerCP was not really bad, but not really > great either. What is your experience?? Was unable find any > systematic comparison in the literature that looked at PE-Cy7 or > APC-Cy7. Does anyone have good validation data? > > 2) Alternatively, I can buy CD133 biotin and label it with FITC. > > Does anyone have good experience with that approach? What can I > expect with regard to signal strength, background, and prep hassle? > > > > Your advise is very much appreciated! > > > > Kind wishes, > > > > Jos > > > > Jos A. Bosch, PhD > > Lecturer in Exercise and Behavioural Immunology > > School of Sport and Exercise Sciences > > University of Birmingham > > Birmingham B15 2TT > > United Kingdom > > > > T: (+44) 0121 4148105 > > F: (+44) 0121 4144121 > > http://www.sportex.bham.ac.uk/staff/boschja > > > >Received on Sun Dec 23 15:58:00 2007
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