Human peripheral blood mononuclear cells (PBMCs) were separated by Ficoll-Paque (GE Health Care, Piscataway, NJ) gradient centrifugation. CD14+ cells were isolated from PBMCs by direct positive isolation using Dynabeads CD14 (Thermo Fisher Scientific) according to the manufacturer’s instructions. CD14+ cells were cultured in complete medium [RPMI 1640, 10% fetal calf serum, 5% human AB serum, 10 mM HEPES, 1% Penicillin-Streptomycin, 10 ng mL−1 macrophage colony-stimulating factor (Millipore, Billerica, MA)] for 7 days for differentiation into MDMs [24 (link)]. This study was approved by the University at Buffalo’s Institutional Review Board (IRB) committee, and the procedures followed are in accordance with institutional guidelines.
Protocol detail
Differentiation of Human Monocytes to Macrophages
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Buffalo
Cells
Centrifugation
Cultured medium
Fetal calf serum
Ficoll
Hepes
Human
Isolation
Macrophage colony stimulating factor
Mdms
Penicillin
Peripheral blood mononuclear cells
Serum
Streptomycin
Corresponding Organization :
Other organizations : University at Buffalo, State University of New York, University of the Western Cape
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Variable analysis
independent variables
- Isolation of CD14+ cells from PBMCs using Dynabeads CD14
- Differentiation of CD14+ cells into MDMs by culturing in complete medium for 7 days
- Differentiation of CD14+ cells into MDMs
- RPMI 1640 medium
- 10% fetal calf serum
- 5% human AB serum
- 10 mM HEPES
- 1% Penicillin-Streptomycin
- 10 ng mL−1 macrophage colony-stimulating factor
- Positive control: None mentioned
- Negative control: None mentioned
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