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Rapid method for isolation of normal human peripheral blood eosinophils on
discontinuous Percoll gradients and comparison with neutrophils
RL Roberts and JI Gallin
Previous studies on human eosinophils often have used cells from patients
with hypereosinophilia syndrome or parasitosis owing to the difficulty in
isolating pure populations of eosinophils from normal individuals. In the
present study, human eosinophils were isolated with a purity of 97%, with
70% recovery from normal individuals with blood eosinophil counts of less
than 3%. Human eosinophils are denser than neutrophils, but the range of
densities of the two cell types overlap, making purification of eosinophils
by density-gradient centrifugation difficult. However, if neutrophils were
exposed to the chemotactic peptide (f-Met-Leu-Phe), which did not stimulate
eosinophils, the neutrophils' density decreased, shifting them away from
the density of eosinophils. Whole normal blood anticoagulated with EDTA was
incubated at 37 degrees C for 15 minutes with 10(-6) mol/L f-Met-Leu-Phe
and then layered over a discontinuous Percoll gradient (65% and 75% in
diluted phosphate-buffered saline) and centrifuged at 400 g for 25 minutes
at 22 degrees C. The cell layer between the 65% and 75% Percoll was
collected and washed, and hypotonic lysis was used to remove erythrocytes.
This cell layer contained 97.3 +/- 0.7% eosinophils (N = 8) with a yield of
4.9 X 10(4) eosinophils per milliliter of whole blood, or 70% of the total
eosinophil count. The isolated eosinophils were in a quiescent state but
responded to Escherichia coli endotoxin- activated serum with shape change
and chemotaxis, membrane depolarization, and reduced nitroblue tetrazolium
(96.0 +/- 1.0%), when stimulated with phorbol myristate acetate. In
phagocytic assays, 89.3 +/- 1.3% of the eosinophils ingested Candida
albicans v 96.0% +/- 1.0% of neutrophils. In contrast, the eosinophils did
not respond chemotactically, alter membrane potential, or reduce nitroblue
tetrazolium when treated with f-Met-Leu-Phe, and studies with f-Met-Leu-
[3H]Phe showed that normal eosinophils lacked expression of receptors for
f-Met-Leu-Phe. In control studies, normal eosinophils that were not exposed
to f-Met-Leu-Phe during purification also failed to respond to
f-Met-Leu-Phe, indicating intrinsic differences between normal eosinophils
and neutrophils. Thus, exposure of whole blood to f-Met-Leu- Phe, followed
by separation on Percoll is a simple method for rapid isolation of normal
human eosinophils.
Volume 65,
Issue 2,
pp. 433-440,
02/01/1985
Copyright © 1985 by The American Society of Hematology
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