Peripheral blood was collected from volunteers, and neutrophils and monocytes were isolated using a modified standard protocol.16 (link) Briefly, whole blood containing acid citrate dextrose as the anti-coagulant was mixed with 10% dextran (MP Biomedicals, Santa Ana, California, USA) to allow for red blood cell sedimentation. The upper layer was removed and overlayed on Ficoll Paque-PLUS (GE Healthcare, Little Chalfont, UK) and centrifuged at 490×g for 10 min. The peripheral blood mononuclear cells at the interface of plasma and Ficoll were isolated, and monocytes were positively selected using magnetic beads against CD14. From the granulocyte pellet, neutrophils were positively selected using magnetic beads against CD16. Further details are provided in the online supplement . Typical purity of both cell types were 99% or greater.
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Isolation of Neutrophils and Monocytes from Peripheral Blood
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Acid citrate dextrose
Blood
Blood plasma cells
Cell types
Coagulant
Dextran
Ficoll
Granulocyte
Monocytes
Neutrophils
Red blood cell sedimentation
Supplement
Volunteers
Corresponding Organization : Woolcock Institute of Medical Research
Other organizations : University of Newcastle Australia, University of Sydney, University Medical Center Groningen, University of Groningen, University of Technology Sydney
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Variable analysis
independent variables
- Isolation of neutrophils and monocytes from peripheral blood using a modified standard protocol
- Purity of the isolated neutrophils and monocytes
- Whole blood containing acid citrate dextrose as the anti-coagulant
- Dextran (MP Biomedicals, Santa Ana, California, USA) for red blood cell sedimentation
- Ficoll Paque-PLUS (GE Healthcare, Little Chalfont, UK) for centrifugation
- Magnetic beads against CD14 for positive selection of monocytes
- Magnetic beads against CD16 for positive selection of neutrophils
- Positive control: Not explicitly mentioned
- Negative control: Not explicitly mentioned
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