Human monocyte isolation methods influence cytokine production from in vitro generated dendritic cells
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Updated: Aug 19, 2026
Characterization of Human Monocyte-derived Dendritic Cells by Imaging Flow Cytometry: A Comparison between Two Monocyte Isolation Protocols
Published on: October 18, 2016
Eyad Elkord1, Paul E Williams, Howard Kynaston
1Department of Medical Biochemistry and Immunology, University of Wales College of Medicine, Cardiff, UK. elkordei@cardiff.ac.uk
Abstract:
There is growing interest in the in vitro generation of dendritic cells (DC) from peripheral blood monocytes, but the effect of the method chosen to isolate CD14+ monocytes for subsequent DC generation is poorly documented. The method used to isolate monocytes may have an impact on the subsequent function of DC by affecting their ability to express costimulatory molecules (CD80/86), maturation marker (CD83) and/or to produce important immunomodulatory cytokines. In this study, we show that the positive selection of monocytes by anti-CD14-coated microbeads inhibits the lipopolysaccharide (LPS)-induced production of interleukin (IL)-12, IL-10 and tumour necrosis factor-alpha (TNF-alpha) from human DC. However, when DC were grown from monocytes isolated by plastic adherence, LPS induced the production of much higher levels of these cytokines. DC derived from adherence-isolated monocytes induced the development of potent cytotoxic T lymphocytes of the Tc1 subset specific for influenza matrix protein, as confirmed by interferon-gamma (IFN-gamma) enzyme-linked immunosorbent spot-forming cell assay (ELISPOT), cytotoxicity assay, major histocompatibility complex (MHC)-peptide tetrameric complexes and T helper 1/T helper 2 (Th1/Th2) cytokine production assays.
Insights
Monocyte isolation methods impact dendritic cell (DC) function. Plastic adherence yields DCs with superior cytokine production and T-cell activation compared to microbead selection, crucial for immunotherapy research.
Area of Science:
- Immunology
- Cell Biology
- Biotechnology
Background:
- Generating dendritic cells (DCs) in vitro from peripheral blood monocytes is of increasing interest.
- The method used to isolate monocytes may affect DC function, including costimulatory molecule expression and cytokine production.
Purpose of the Study:
- To investigate the impact of different monocyte isolation methods on human dendritic cell generation and function.
- To compare the immunomodulatory capacity of DCs derived from monocytes isolated by positive selection versus plastic adherence.
Main Methods:
- Monocytes were isolated using anti-CD14-coated microbeads (positive selection) or plastic adherence.
- Dendritic cells were generated in vitro.
- Lipopolysaccharide (LPS)-induced cytokine production (IL-12, IL-10, TNF-alpha) was measured.
- T-cell responses, including cytotoxic T lymphocyte (CTL) induction and cytokine profiles, were assessed.
Main Results:
- Positive selection of monocytes using anti-CD14 microbeads inhibited LPS-induced production of IL-12, IL-10, and TNF-alpha by DCs.
- DCs generated from plastic-adherent monocytes exhibited significantly higher LPS-induced cytokine production.
- DCs derived from adherence-isolated monocytes effectively induced potent Tc1 cytotoxic T lymphocytes specific for influenza matrix protein.
Conclusions:
- Monocyte isolation method critically influences DC function and cytokine production.
- Plastic adherence is a preferred method for generating DCs with enhanced immunomodulatory capabilities for potential therapeutic applications.
- The choice of monocyte isolation technique impacts the ability of derived DCs to stimulate robust T-cell responses.
