Hi to all, Thanks for some very good ideas about protocols and products to try. Mouse blood is harder to work with because the nucleated RBCs do not lyse and they can be a sustantial population. We are trying to go back to a separation gradient method as the lysis buffers seem to give us too much non-specific antibody binding, so maybe the cell membranes are damaged in some way. We are trying to gate for the CD45 (-) population and then look for circulating endothelial cells within that group. This is a very small population, so when we were getting too many cells bound to either VEGF or CD117 (progenitor) antibodies, we knew something is amiss. The Ficoll-Paque product we were using has a density of 1.077 and they now have a product (PREMIUM) that is 1.084, which may be better. I was told we could also use PERGOLL which can be adusted to densities from 1.023 up to about 1.09+, alternatively, we may try the Cedarlane product recommended by several posters to this list. Hopefully, this will leave us with sufficient cells that bind correctly! Any other ideas will be greatly appreciated. Lori -- Lori Rice, Ph.D. University of Florida lrice@ufl.eduReceived on Thu Feb 14 13:38:00 2008
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