Purification of human basophils and mast cells by multistep separation technique and mAb to CDw17 and CD117/c-kit.
Basophils and mast cells represent distinct cell lineages within the hemopoietic 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 granulocytes from the peripheral blood and of mast cells from human dispersed tissues. Basophils (n = 9) were purified by current counterflow elutriation followed by depletion of monocytes with CD14 mAb conjugated to magnetic beads, and subsequent cell sorting for CD217+ 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 digestion followed by current counterflow elutriation and sorting with CD117/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 histamine on induction with IgE plus anti-IgE. Furthermore, the PCR technique could be applied on pure cells and confirmed expression of high affinity IgE receptor (Fc epsilon R1) alpha chain mRNA. Thus, by combining isolation techniques including elutriation, magnetic cell depletion and cell sorting with mAb, functionally intact normal human basophils and mast cells can be enriched to homogeneity.
Willheim
M
M
Institute of General and Experimental Pathology, University of Vienna, Neubau AKH, Austria.
Agis
H
H
Sperr
W R
WR
Köller
M
M
Bankl
H C
HC
Kiener
H
H
Fritsch
G
G
Füreder
W
W
Spittler
A
A
Graninger
W
W
Scheiner
O
O
Gadner
H
H
Lechner
K
K
Boltz-Nitulescu
G
G
Valent
P
P
D000911:Antibodies, Monoclonal / Q000276:immunology; D015703:Antigens, CD / Q000276:immunology*; D001483:Base Sequence; D001491:Basophils / Q000166:cytology / Q000276:immunology*; D002469:Cell Separation / Q000379:methods*; D002478:Cells, Cultured; D005434:Flow Cytometry; D006636:Histamine Release; D006801:Humans; D007073:Immunoglobulin E / Q000276:immunology; D008407:Mast Cells / Q000166:cytology / Q000276:immunology*; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction; D011518:Proto-Oncogene Proteins / Q000276:immunology; D019009:Proto-Oncogene Proteins c-kit; D020794:Receptor Protein-Tyrosine Kinases / Q000276:immunology; D016184:Receptors, Colony-Stimulating Factor / Q000276:immunology; D017455:Receptors, IgE / Q000235:genetics; D014461:Ultracentrifugation / Q000379:methods
D000911:Antibodies, Monoclonal; D015703:Antigens, CD; D011518:Proto-Oncogene Proteins; D016184:Receptors, Colony-Stimulating Factor; D017455:Receptors, IgE; D007073:Immunoglobulin E; D019009:Proto-Oncogene Proteins c-kit; D020794:Receptor Protein-Tyrosine Kinases