[Cytometry] congenic CD45.1/.2 blocking

Andy Hoffman andrew.hoffman at tufts.edu
Fri May 8 16:22:11 EDT 2009


We're doing a congenic mouse study transplanting directly sorted lung 
cells from CD45.2 mice into lungs of CD45.1 mice.   We have noted that 
the anti-CD45.1 antibody reacts with CD45.2 cells and vice versa 
although the intensity is 1 log lower when mismatched.    I apologize - 
I realize this has come up before on the listserve, but I was wondering 
if someone could suggest an optimal blocking procedure for such an 
experiment, or perhaps comment whether we should 'expect' a certain 
level of cross-reactivity even in the best circumstances.  For blocking 
the FcR we use anti-CD16/32 (eBio or BD) or mouse polyclonal serum (10 
min at 4oC) immediately before introducing the directly conjugated 
anti-CD45 (.1 or .2 or both) antibody.   Our staining buffer is media 
(DMEM:F12 + 10% FBS).  Also is there a problem with mixing the two 
antibodies (antiCD45.1 and anti-CD45.2)  in the same sample of cells, 
since this would be imperative to understand our findings in the 
transplanted recipients.

Thanks!
Andy

-- 
Andrew M. Hoffman, D.V.M., D.V.Sc., 
Diplomate, A.C.V.I.M. (Large Animal Medicine)
Associate Professor 
Cummings School of Veterinary Medicine
Tufts University
Director, Lung Function Testing Laboratory
Department of Clinical Sciences
Bldg 21, Suite 110
200 Westboro Road
North Grafton, MA 01536
andrew.hoffman at tufts.edu
Phone (508) 887 4589
Fax   (508) 887 4590



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