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A method is described for the isolation of highly purified peritoneal eosinophils from the polymyxin B-treated guinea pigs on density gradients ofNycodenz, a new non-iodinated gradient medium. After more than 8 weeks ofpolymyxin B injection into the peritoneal cavity, eosinophils comprized about 50% of the exudative cells (1 x 10“ cells/animal). The rest of the collected cells were composed of mononuclear cells but no neutrophils were found. Four visible cell bands were fractionated by centrifugation through Nycodenz solutions. Eosinophils more than 98% pure were recovered at the density interface between 1.088 and 1.098 glml (band 4). The recovery rate was 45 to 80°/0. This separation technique did not affect the morphology, viability or metabolic capacities of the cells. Most techniques for in vitro studies of physiological and biochemical composition of eosinophils require not only purifying adequate numbers of the cells but also collecting the cells with intact metabolic activities. Usually, eosinophils stimulated by pathogenic conditions, such as patients or animals with eosinophilia, are used in an attempt to ascertain normal eosinophil physiology. Thus, Beeson and Bass (2) reviewed the literature concerning the contradiction of using ‘pathological’ eosinophils in the investigation of ‘normal’ eosinophils. Recent studies using density gradient centrifugation of peripheral blood leucocytes have shown that eosinophils from patients with hyper-eosinophilic syndrome and some parasitic diseases are less dense than those from healthy persons (5, 16, 17, 22). In addition, these hypodense eosinophils showed morphological, functional, and metabolic changes (5, 16, 17, 22). In this communication, we describe an isolation method of r I. highly purified peritoneal eosinophils with normal structure and metabolic activities from polymyxin B-treated guinea pigs on newly introduced density gradients of Nycodenz. These isolated cells are expected to be valuable in studies of biological properties of the eosinophil leucocytes under the pathogenic conditions. MATERIALS AND METHODS Preparation of Eosinophil-rich Peritoneal Cells in Guinea Pigs Guinea-pig peritoneal eosinophil-rich exudates were prepared by weekly injection to Hartley strain guinea pigs with 5,000 units/ml of polymyxin B sulfate (Pfizer) for at least 8 weeks after a modification of the method of Pincus (15). Peritoneal cells were collected in Hanks’ balanced salt solution (Hanks’ BSS) containing 20 units/ml of sodium heparin on the next day after the last injection. The cells were centrifuged (100 g), washed twice and

biomedical research

&lt;b&gt;ISOLATION OF PERITONEAL EOSINOPHILS FROM POLYMYXIN B-TREATED GUINEA PIGS ON DENSITY GRADIENTS OF NYCODENZ, A NEW NON-IONIC IODINATED GRADIENT &lt;/b&gt;&lt;b&gt;MEDIUM &lt;/b&gt;