We are using TER119-PE from Pharmingen to stain mouse erythroid cells. This is one antibody of many that we are using to study lineage repopulation capacity of transplanted cells. TER119 antibody recognizes mouse erythrocytes and should also recognize any nucleated red cells present in the blood. Normally we do an RBC lysis prior to staining since this should not lyse the nucleated cells which are the cells we are interested in. The problem is that we are seeing nonspecific staining of other non erythroid cells with TER119 as evidenced by two color staining with other markers for B,T, and myeloid cells. All other antibodies we use stain clean and specific. We always pretreat the cells with Fc block from Pharmingen to reduce nonspecific binding, and we always compare our staining to an isotype matched control. A titer of TER119 showed the same titer curves for binding to RBCs and non erythroid cells which suggested that this nonspecific staining was not some low affinity binding. Staining was then compared on non-RBC lysed cells, lysed cells, and mononuclear cells harvested from a 1.077 ficoll-hypaque density gradient. Nonspecific staining of non erythroid cells was very low on cells from the Ficoll gradient, was terrible on non lysed cells, and was intermediate on RBC lysed cells. All this led me to think that TER119 may be staining RBCs bound to nucleated cells. We do our staining with PBS/2%Fetal bovine serum. PI is used to mark dead cells which are very few in these samples. Does this sound like the likely reason? Is there something I could change to eliminate this problem? All suggestions appreciated. I am wondering if there is something I can do to avoid the Ficoll since cell numbers for staining are limited and we also want to assess the presence of mature granulocytes. The Ficoll would have to be used only for cells stained for TER and that would be painful! Thanks! Dave Flowers Program in Pediatric Oncology Fred Hutchinson Cancer Research Center Seattle, WA, USA
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