A protocol to isolate and characterize pure monocytes and generate monocyte-derived dendritic cells through FBS-Coated flasks
Maryam Meskini, Amir Amanzadeh, Fahimeh Salehi, Saeid Bouzari, Morteza Karimipoor, Andrea Fuso, Abolfazl Fateh, Seyed Davar Siadat
Abstract
This study explores methods to isolate high-pure monocytes and optimize the best growth factor concentration to generate monocytes-derived dendritic cells (mo-DCs), subset DC1, which is crucial in immune responses. Three protocols for monocyte isolation from peripheral blood mononuclear cells (PBMCs) were evaluated: three-hour incubation on FBS-coated flasks; an overnight incubation on FBS-coated flasks; and Magnetic Activated Cell Sorting (MACS). Additionally, five different concentrations of human recombinant granulocyte-macrophage colony-stimulating factor (hrGM-CSF) and human recombinant interleukin-4 (hrIL-4) were compared. We used Flow cytometry to assess the isolation, purification, and generation of pure monocytes characterized as CD14 + , and expression of mo-DC classical markers (HLA-DR, CD80, CD83, and CD86). The obtained results show that monocytes isolated with the second method (overnight incubation) had the highest purity ( P < 0.0001) but the lowest yield ( P > 0.05), balancing purity and cost-effectiveness. A combination of hrGM-CSF and hrIL-4 at 400 U/mL produced the most favorable outcomes, leading to the highest rate of mo-DC generation ( P < 0.05). Notably, this concentration resulted in increasing expression of HLA-DR, CD80, and CD86 surface markers in the generated DCs ( P < 0.0001), with no changes in CD83 expression levels. In conclusion, this study offers valuable insights into selecting the optimal approach for monocyte isolation and mo-DC generation in various research contexts, providing a foundation for more effective immunological studies.