An improved method for the purification of basophilic granulocytes from human blood.
Studies on human basophils are hampered by the low number of basophils in peripheral blood. Here we describe the purification of human basophilic granulocytes with immunomagnetic beads to improve an already established method of purification. A 70% pure basophil suspension was incubated with monoclonal antibodies (CD2, CD14, CD16 and CD19) recognizing the contaminating cells. After incubation with magnetic beads coated with goat anti-mouse IgG, the bead-cell rosettes were removed by a magnet. In this way, the basophil purity increased to 94%. The loss of basophils during the bead procedure was about 20%. The amount of histamine per basophil and the spontaneous release of histamine during subsequent incubation of the cells was not altered by the purification procedure. The kinetics and the dose response of histamine release after the addition of anti-IgE or FMLP was also unchanged. Binding of CD63 was not altered, indicating that the purification procedure did not result in activation of the basophils. This improved method for the purification of human basophils should permit the measurement of non-basophil-specific parameters, such as changes in intracellular free Ca2+, without the problem of interference from contaminating cells.
Mul
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FP
Central Laboratory of the Netherlands Red Cross Blood Transfusion Service, Amsterdam.
Knol
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EF
Roos
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D000911:Antibodies, Monoclonal; D001491:Basophils / Q000276:immunology / Q000378:metabolism; D002469:Cell Separation; D002498:Centrifugation; D005434:Flow Cytometry; D005455:Fluorescent Antibody Technique; D006636:Histamine Release; D006801:Humans; D007073:Immunoglobulin E / Q000032:analysis; D011948:Receptors, Antigen, T-Cell / Q000032:analysis
D000911:Antibodies, Monoclonal; D011948:Receptors, Antigen, T-Cell; D007073:Immunoglobulin E