We find that normal hypaque-ficoll freshly isolated (unculturd) PBl from heparinized whole blood show some degree of apoptosis. Gerry- We also find base-line apoptosis in F/H isolated normal PBMC using Annexin V binding. We found that this is reduced if we use Cellsep (Larex Inc., St. Paul, MN) separation medium. F/H is not entirely non-toxic. You can usually see a decrease in forward scatter after F/H. I suspect that the polysucrose exerts quite an osmotic stress. Cellsep is a food grade (I'm not making this up) polysaccharide (arabinogalactan) and appears to be a much gentler separation medium. Recovery of lymphocytes is similar to or better than F/H, and platelets do not coseparate with lymphocytes. Scatter properties are retained and Annexin V binding is lower in normal PBMC. More importantly, freshly isolated lymphocytes undergoing early apoptosis may not function normally with respect to osmotic regulation. We find that we preferentially lose early apoptotic lymphocytes through the F/H gradient. Thus PBMC from early stem cell transplant recipients (who have high spontaneous apoptosis) give lower apoptosis when preseparated on F/H. Summary: I think F/H induces apoptosis in non-apoptotic cells and causes loss of early apoptotic cells. Albert D. Donnenberg, Ph.D. Co-director Hematopoiesis & Hematologic Malignancies Program University of Pittsburgh Cancer Institute (412) 624-9596
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