We would like to do indirect BAT (ie incubating basophils with serum from allergic or sensitized patients) using a source of cells that can last awhile. After obtaining buffy coats which we Ficoll treated, we separately froze both the PBMC and the granulocytes, as from the literature we really could not figure out the best are to look. While we saw a CD123+ HLADR- population from the pre-frozen cells, which elevated surface CD63 in response to anti IgE stimulation, we could not find a CD123+ HLADR- population after thawing (neither the PBMC nor granulocytes). We tried growing in the presence of IL3 for a few days, but though the cells seemed viable, we could not see anything (at least based on these markers).
Any other suggestions? Might it be reasonable to try other sources such as cord blood, or even a cell line like KU812F ? I should note that we are not necessarily looking for high purity, as long as we have enough cells, even the 0.5-1% that is in blood might be workable.
Thank you in advance!
One can use the CD103 that binds to blood basophils when activated: FACS must compare the marking before and after activation to obtain the percentage of basophils.
The antibody 212H6 is a good marker of basophils. This antibody is specific for blood basophils, which works by FACS and which moreover do not porvoque basophil degranulation.
This antibody is recommended to isolate basophils without induced histamine release by the selection, for use in the degranulation assays.
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