A rapid simple method of basophil purification by density centrifugation on Percoll.

Abstract

This study describes simple and relatively rapid methods of purifying basophils from human peripheral blood. Our initial studies defined the densities of basophils in several donors and helped develop strategies for selectively concentrating basophils by isopyknic centrifugation over Percoll. We layered anticoagulated blood over Percoll of appropriate density, centrifuged these for 10 min, and collected the cells held in Percoll separately from the overlying band of mononuclear cells. Most of the basophils were distributed between densities of 1.072 and 1.078 g/ml, with a peak at approximately 1.074 to 1.076 g/ml. Basophil purity of 24.4 +/- 4% (range 5.6 to 50%) was achieved in a Percoll gradient of 1.076 g/ml due to both the increased numbers of basophils and the low numbers of mononuclear cells at this gradient. The cells isolated by this method are viable, appear morphologically normal, and release histamine to various stimuli in a normal manner. A second centrifugation of the cells obtained from this density over appropriate discontinuous gradients of Percoll leads to further enhancement of basophil purity. The main limitation of this technique appears to be the relatively low yield (percent of recovery) of the basophil (less than or equal to 20% for the two-step procedures).