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Purification of human blood eosinophils by a combination method using anti-CD16 monoclonal antibody, immunobeads, and
M Miyasato1, S Tsuda, N Kitamura
1Department of Dermatology, Kurume University School of Medicine, Japan.
Insights
This study presents a simple method to isolate human blood eosinophil phenotypes using anti-CD16 antibody and Nycodenz density gradients. The technique effectively separates normodense and hypodense eosinophils for further immunological studies.
Area of Science:
- Immunology
- Cell Biology
Background:
- Eosinophils play crucial roles in immune responses, particularly in parasitic infections and allergic reactions.
- Accurate isolation of eosinophil phenotypes is essential for understanding their functions and developing targeted therapies.
Purpose of the Study:
- To develop a straightforward and efficient method for purifying human blood eosinophil phenotypes.
- To enable the simultaneous isolation of both normodense and hypodense eosinophil subpopulations.
Main Methods:
- Utilized a combination of anti-CD16 monoclonal antibody, immunobeads, and Nycodenz density gradient centrifugation.
- Employed sequential cell separation steps including density gradient, plastic adherence, and immunobead-based purification.
- Isolated eosinophil phenotypes based on their differential binding and density properties.
Main Results:
- Successfully procured purified human blood eosinophil phenotypes.
- Achieved simultaneous preparation of highly purified normodense (> 1.080 g/ml) and hypodense (< 1.080 g/ml) eosinophils.
- Isolated eosinophils demonstrated functional responses, including chemiluminescence to opsonized zymosans and helminthotoxic activity.
Conclusions:
- The described method provides a simple and effective means for isolating eosinophil phenotypes from human blood.
- This technique allows for the simultaneous study of normodense and hypodense eosinophils, facilitating research into their distinct immunological properties.
Abstract:
A simple method is described for the procurement of human blood eosinophil phenotypes by combining an anti-CD16 monoclonal antibody, immunobeads, and a non-toxic and non-ionic density gradient medium, Nycodenz. The purification depends on the removal of mononuclear cells using a 1.076/1.102 g/ml Nycodenz density gradient, partial removal of neutrophils based on different binding to plastic dishes, interaction of residual neutrophils with immunobeads via an anti-CD16 monoclonal antibody and, finally, extraction of eosinophil phenotypes by sifting the immunobeads-loaded neutrophils through an 1.080/1.102 g/ml Nycodenz density gradient. This method permits simultaneous preparation of highly purified normodense (> 1.080 g/ml) and hypodense eosinophils (< 1.080 g/ml) with reasonable chemiluminescence responses to opsonized zymosans and helminthotoxic activity to opsonized schistosomula corresponding to their own immunocytological properties.
