Human peripheral blood mononuclear cells were isolated from fresh blood leukocyte source packs (American Red Cross, Columbus, OH) by density gradient centrifugation using a Ficoll-Hypaque density gradient (GE Healthcare, formerly Amersham Biosciences, Piscataway, NJ). Positive selection for monocytes was performed using CD14 antibody conjugated to magnetic beads (Miltenyi Biotec, Auburn, CA). Purity of these preparations of monocytes was >90% as determined by fluorescence-activated cell sorting analyses using CD14 antibodies. Differentiation of these cells to macrophages (MDM) was performed as described (29 (link)).
Protocol detail
Isolation and Differentiation of Monocyte-Derived Macrophages
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Antibodies
Antibody
Blood
Cells differentiation
Density gradient centrifugation
Ficoll
Human
Hypaque
Leukocyte
Macrophages
Monocytes
Peripheral blood mononuclear cells
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Variable analysis
independent variables
- Density gradient centrifugation using Ficoll-Hypaque
- Positive selection for monocytes using CD14 antibody conjugated to magnetic beads
- Purity of monocyte preparations (>90% as determined by fluorescence-activated cell sorting analyses using CD14 antibodies)
- Differentiation of monocytes to macrophages (MDM)
- Fresh blood leukocyte source packs (American Red Cross, Columbus, OH)
- Positive control: Not explicitly mentioned
- Negative control: Not explicitly mentioned
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