PURIFICATION OF HUMAN BASOPHILS AND MAST-CELLS BY MULTISTEP SEPARATION TECHNIQUE AND MAB TO CDWL7 AND CD117 C-KIT/

Basophils and mast cells represent distinct cell lineages within the h emopoietic system. Based on the unique cell surface antigen profile of both cells, we have established methods which allow the reproducible purification to homogeneity (> 99%) of normal human basophil granulocy tes from the peripheral brood and of mast cells from human dispersed t issues. Basophils (n = 9) were purified by current counterflow elutria tion followed by depletion of monocytes with CD14 mAb conjugated to ma gnetic beads, and subsequent cell sorting for CDw17(+) cells. Basophil purity was 99.5 +/- 0.4% (range 98.7-99.9%). Mast cells were obtained from lung (n = 6), uterus (n = 1), mastocytosis bone marrow (n = 2), and human foreskin (n = 2). Mast cells were purified by collagenase di gestion followed by current counterflow elutriation and sorting with C D117/c-kit mAb. Mast cell purity was 99.4 +/- 0.7% (range: 97.5-99.9%) . Purified cells were more than 90% viable and were able to release hi stamine on induction with IgE plus anti-IgE. Furthermore, the PCR tech nique could be applied on pure cells and confirmed expression of high affinity IgE receptor (Fc epsilon R1) alpha chain mRNA. Thus, by combi ning isolation techniques including elutriation, magnetic cell depleti on and cell sorting with mAb, functionally intact normal human basophi ls and mast cells can be enriched to homogeneity.