Purification of human basophils by negative selection.
Enriched populations of human basophils were prepared using a combination of negative selection techniques. First, a 2-step discontinuous gradient was made using 65% and 55% isotonic Percoll. Purification of plasma basophils by centrifugation through the gradient increased basophils from 0.96 +/- 0.55% to 14.6 +/- 7.9% (n = 9). In 5 other experiments a second step was added in which contaminating mononuclear cells were removed using a panning technique. In this technique, Percoll separated cells were treated with anti-T-cell antibodies. Contaminating T-cells were selectively removed by adherence to a petri dish coated with affinity purified goat anti-mouse IgG. Basophil purity was increased to 34 +/- 15% using this step. These experiments demonstrated that negative selection techniques can yield an increase in basophil purity 50-100-fold. The purified basophils contained the expected quantity of histamine (1.36-1.54 pg/basophil) and released the same amount of histamine in response response to different concentrations of goat anti-human IgE Fc as did unpurified cells.
D000911:Antibodies, Monoclonal; D001491:Basophils; D002469:Cell Separation / Q000379:methods*; D002500:Centrifugation, Isopycnic / Q000379:methods; D006636:Histamine Release; D006801:Humans; D011205:Povidone; D012822:Silicon Dioxide
D000911:Antibodies, Monoclonal; C016039:Percoll; D012822:Silicon Dioxide; D011205:Povidone