JoVE Logo
Faculty Resource Center

Sign In





Representative Results





Immunology and Infection

Characterization of Human Monocyte-derived Dendritic Cells by Imaging Flow Cytometry: A Comparison between Two Monocyte Isolation Protocols

Published: October 18th, 2016



1Department of Immunology, Herbert Wertheim College of Medicine, Florida International University, 2Millipore Sigma

This study compares two different methods of human monocyte isolation for obtaining in vitro dendritic cells (DCs). Monocytes are selected by adherence or negatively enriched by magnetic separation. Monocyte yield and viability along with MDDC viability, proliferation and CD11c/CD14 surface marker expression will be compared between both methods.

Dendritic cells (DCs) are antigen presenting cells of the immune system that play a crucial role in lymphocyte responses, host defense mechanisms, and pathogenesis of inflammation. Isolation and study of DCs have been important in biological research because of their distinctive features. Although they are essential key mediators of the immune system, DCs are very rare in blood, accounting for approximately 0.1 - 1% of total blood mononuclear cells. Therefore, alternatives for isolation methods rely on the differentiation of DCs from monocytes isolated from peripheral blood mononuclear cells (PBMCs). The utilization of proper isolation techniques that combine simplicity, affordability, high purity, and high yield of cells is imperative to consider. In the current study, two distinct methods for the generation of DCs will be compared. Monocytes were selected by adherence or negatively enriched using magnetic separation procedure followed by differentiation into DCs with IL-4 and GM-CSF. Monocyte and MDDC viability, proliferation, and phenotype were assessed using viability dyes, MTT assay, and CD11c/ CD14 surface marker analysis by imaging flow cytometry. Although the magnetic separation method yielded a significant higher percentage of monocytes with higher proliferative capacity when compared to the adhesion method, the findings have demonstrated the ability of both techniques to simultaneously generate monocytes that are capable of proliferating and differentiating into viable CD11c+ MDDCs after seven days in culture. Both methods yielded > 70% CD11c+ MDDCs. Therefore, our results provide insights that contribute to the development of reliable methods for isolation and characterization of human DCs.

Dendritic cells (DCs) are essential mediators of the innate and adaptive immune systems. They function to induce primary immune responses and facilitate the development of immunological memory. These cells are primarily responsible for antigen capture, migration and T cell stimulation and are therefore referred to as professional antigen presenting cells (APCs) 1.Manipulation of DCs could be utilized across a wide variety of research fields and in the clinical setting to treat different inflammatory diseases such as HIV 6,7, cancer 8, autoimmune diseases 9, and allergic responses 10. DCs are also being used for su....

Log in or to access full content. Learn more about your institution’s access to JoVE content here

Overall human blood studies have been reviewed and approved by the Institutional Review Board (IRB) of FIU, IRB protocol approval # IRB-13-0440. Human leukopaks were purchased from the community blood bank in Miami, FL.

1. Isolation of PBMCs by Standard Density Gradient Technique

  1. Perform a 1:1 dilution of blood with 1x-phosphate-buffered saline in a T75 flask.
  2. Pipette 15 ml of density gradient solution into 50 ml centrifuge tubes and carefully layer (25 - 30 ml/tube) of t.......

Log in or to access full content. Learn more about your institution’s access to JoVE content here

Monocyte Yield by Magnetic Separation is Higher Compared to Monocyte Yield by Adherence Method

Data presented in Figure 1 display PBMC and monocyte cell counts by the trypan blue exclusion method at the day of isolation of PBMCs and separation of monocytes. On average, monocytes isolated by the adherence method accounted for approximately 6.2 percent of total PBMCs while monocytes isolated by ma.......

Log in or to access full content. Learn more about your institution’s access to JoVE content here

Based on the known difficulties of isolating and generating MDDCs from human blood, the present study aimed to provide a comprehensive comparison of two well-established methods for the generation of MDDCs. The first method compared is a well-established traditional method for generating MDDCs by exploiting the ability of monocytes to adhere to glass or plastic (adherence method) 21,22,27. The adherence method is fast and cost effective, and does not require the use of complex equipment. However, some disadvan.......

Log in or to access full content. Learn more about your institution’s access to JoVE content here

This research is supported by the National Institute on Alcohol Abuse and Alcoholism, award K99/R00 AA021264. Additional lab support as part of startup package has been received from the Department of Immunology, Institute of NeuroImmune Pharmacology, Herbert Wertheim College of Medicine, and FIU- Office of Research and Economic Development. Gianna Casteleiro was supported by NIH/NIGMS R25 GM061347. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health.


Log in or to access full content. Learn more about your institution’s access to JoVE content here

Name Company Catalog Number Comments
Ficoll-Paque GE Healthcare 17-5442-03 Must be used at room temperature
Phosphate-buffered saline (PBS) Life Technologies 10010-023
ACK Lysing buffer Quality Biological 118-156-101
RPMI 1640 medium Life Technologies 22400-089
Antibiotic-Antimycotic (100X) Life Technologies 15240-062
Fetal Bovine Serum (FBS) Life Technologies 16000-044
RoboSep buffer StemCell 20104
EasySep Human monocyte enrichment kit StemCell 19059
TC20 Automated cell counter Bio-Rad 145-0101
FITC-Dextran Sigma Aldrich FD4-100MG
Trypan blue stain (0.4%) Life Technologies 15250-061
Synergy 2 multi-mode reader Biotek 7131000
XTT Sodium salt bioreagent (XTT) Sigma Aldrich X4626-100MG
Dimethyl sulfoxide bioreagent (DMS) Sigma Aldrich D8418-500ML
Thiazolyl blue tetrazolium bromide (MTT) Sigma Aldrich m5655-500MG
Sodium Dodecyl Sulfate (SDS) Bio-Rad 161-0302
Phenazine Methosulfate (DMSO) Sigma Aldrich P9626-1G
Inactivated (HI) Human Serum Chemicon S1-100ML
Accuri C6 Flow Cytometer BD Accuri 653119
FlowSight Amnis Flow Cytometer EMD Millipore 100300

  1. Cella, M., Sallusto, F., Lanzavecchia, A. Origin maturation and antigen presenting function of dendritic cells. Curr Opin Imunol. 9, 10-16 (1997).
  2. Banchereau, J., et al. Immunobiology of Dendritic Cells. Ann Rev Immunol. 18, 767-811 (2000).
  3. Banchereau, J., Steinman, R. M. Dendritic cells and the control of immunity. Nature. 392, 245-252 (1998).
  4. Kaouther, M., Ridha, O. Dendritic Cell-Based Graft Tolerance. ISRN Pharmacol. , (2011).
  5. Steinman, R., Gutchinov, B., Witmer, M., Nussenzweig, M. Dendritic cells are the principal stimulators of the primary mixed leukocyte reaction in mice. J Exp Med. 157, 613-627 (1983).
  6. Nair, M. N., et al. RNAi-directed inhibition of DC-SIGN by dendritic cells: Prospects for HIV-1 therapy. AAPS J. 7, E572-E578 (2005).
  7. Agudelo, M., et al. Chapter 10. Dendritic Cells: Types, Life Cycles and Biological Functions. , 167-177 (2010).
  8. Kajihara, M., Takakura, K., Ohkusa, T., Koido, S. The impact of dendritic cell-tumor fusion cells on cancer vaccines - past progress and future strategies. Immunotherapy. , (2015).
  9. Suwandi, J., Toes, R., Nikolic, T., Roep, B. Inducing tissue specific tolerance in autoimmune disease with tolerogenic dendritic cells. Clin Exp Rheumatol. 33, 0097-0103 (2015).
  10. Gorelik, M., Frischmeyer-Guerrerio, P. A. Innate and adaptive dendritic cell responses to immunotherapy. Curr Opin Allergy Immunol. 15, 575-580 (2015).
  11. Zwolak, A., et al. Peripheral blood dendritic cells in alcoholic and autoimmune liver disorders. Hum Exp Toxicol. 31, 438-446 (2012).
  12. Agudelo, M., et al. Differential expression and functional role of cannabinoid genes in alcohol users. Drug Alcohol Depend. 133, 789-793 (2013).
  13. Nair, M. P., Figueroa, G., Casteleiro, G., Muñoz, K., Agudelo, M. Alcohol Versus Cannabinoids: A Review of Their Opposite Neuro-Immunomodulatory Effects and Future Therapeutic Potentials. J Alcohol Drug Depend. 3, 184 (2015).
  14. Boukli, N. M., et al. Implications of ER Stress, the Unfolded Protein Response, and Pro- and Anti-Apoptotic Protein Fingerprints in Human Monocyte-Derived Dendritic Cells Treated With Alcohol. Alcohol Clin Exp Res. 34, 2081-2088 (2010).
  15. Nair, M. N., Mahajan, S., Sykes, D., Bapardekar, M., Reynolds, J. Methamphetamine Modulates DC-SIGN Expression by Mature Dendritic Cells. J Neuroimmune Pharmacol. 1, 296-304 (2006).
  16. Napuri, J., et al. Cocaine Enhances HIV-1 Infectivity in Monocyte Derived Dendritic Cells by Suppressing microRNA-155. PLoS ONE. 8, e83682 (2013).
  17. Nair, M. P. N., Saiyed, Z. M. Effect of methamphetamine on expression of HIV coreceptors and CC-chemokines by dendritic cells. Life Sciences. 88, 987-994 (2011).
  18. Nair, M. P. N., et al. Cocaine Modulates Dendritic Cell-Specific C Type Intercellular Adhesion Molecule-3-Grabbing Nonintegrin Expression by Dendritic Cells in HIV-1 Patients. J Immunol. 174, 6617-6626 (2005).
  19. Reynolds, J. L., Mahajan, S. D., Sykes, D. E., Schwartz, S. A., Nair, M. P. N. Proteomic analyses of methamphetamine (METH)-induced differential protein expression by immature dendritic cells (IDC). Biochem Biophys Acta. 1774, 433-442 (2007).
  20. Van Voorhis, W., Hair, L., Steinman, R., Kaplan, G. Human dendritic cells. Enrichment and characterization from peripheral blood. J Exp Med. 155, 1172-1187 (1982).
  21. Davis, G. The Mac-1 and p150,95 beta 2 integrins bind denatured proteins to mediate leukocyte cell-substrate adhesion. Exp Cell Res. 200, 242-252 (1992).
  22. Delirezh, N., Shojaeefar, E. Phenotypic and functional comparison between flask adherent and magnetic activated cell sorted monocytes derived dendritic cells. Iran J Immunol. 9, 98-108 (2012).
  23. Lehner, M., Holter, W. Endotoxin-Free Purification of Monocytes for Dendritic Cell Generation via Discontinuous Density Gradient Centrifugation Based on Diluted Ficoll-Paque Plus<sup>®</sup>. Int Arch Allergy Immunol. 128, 73-76 (2002).
  24. Van Brussel, I., et al. Fluorescent activated cell sorting: An effective approach to study dendritic cell subsets in human atherosclerotic plaques. J. Immunol Methods. 417, 76-85 (2015).
  25. Mucci, I., et al. The methodological approach for the generation of humandendritic cells from monocytes affects the maturation state of the resultant dendritic cells. Biologicals. 37, 288-296 (2009).
  26. Yuan, N., et al. . The American Association of Immunologists (AAI). , (2007).
  27. Romani, N., et al. Proliferating dendritic cell progenitors in human blood. J Exp Med. 180, 83-93 (1994).
  28. Bennett, S., Breit, S. N. Variables in the isolation and culture of human monocytes that are of particular relevance to studies of HIV. J Leukoc Biol. 56, 236-240 (1994).
  29. Grützkau, A., Radbruch, A. Small but mighty: How the MACS®-technology based on nanosized superparamagnetic particles has helped to analyze the immune system within the last 20 years. Cytometry Part A. 77A, 643-647 (2010).
  30. El-Sahrigy, S. A., Mohamed, N. A., Talkhan, H. A., Rahman, A. M. A. Comparison between magnetic activated cell sorted monocytes and monocyte adherence techniques for in vitro generation of immature dendritic cells: an Egyptian trial. Cent Eur J Immunol. 40, 18-24 (2015).
  31. Curry, C. V. . Differential Blood Count. , (2015).
  32. Sallusto, F., Lanzavecchia, A. Efficient presentation of soluble antigen by cultured human dendritic cells is maintained by granulocyte/macrophage colony-stimulating factor plus interleukin 4 and downregulated by tumor necrosis factor alpha. J Exp Med. 179, 1109-1118 (1994).
  33. Cavanagh, L. L., Saal, R. J., Grimmett, K. L., Thomas, R. Proliferation in Monocyte-Derived Dendritic Cell Cultures Is Caused by Progenitor Cells Capable of Myeloid Differentiation. Blood. 92, 1598-1607 (1998).
  34. Ardeshna, S. M., et al. Monocyte-derived dendritic cells do not proliferate and are not susceptible to retroviral transduction. Br J Haematol. 108, 817-824 (2000).
  35. Chapuis, F., et al. Differentiation of human dendritic cells from monocytes in vitro. Eur J Immunol. 27, 431-441 (1997).
  36. Zhou, L. J., Tedder, T. F. CD14+ blood monocytes can differentiate into functionally mature CD83+ dendritic cells. Proc Natl Acad Sci. 93, 2588-2592 (1996).
  37. Caux, C., et al. B70/B7-2 is identical to CD86 and is the major functional ligand for CD28 expressed on human dendritic cells. J Exp Med. 180, 1841-1847 (1994).
  38. Caux, C., et al. Activation of human dendritic cells through CD40 cross-linking. J Exp Med. 180, 1263-1272 (1994).
  39. Fujii, S. i., Liu, K., Smith, C., Bonito, A. J., Steinman, R. M. The Linkage of Innate to Adaptive Immunity via Maturing Dendritic Cells In Vivo Requires CD40 Ligation in Addition to Antigen Presentation and CD80/86 Costimulation. J Exp Med. 199, 1607-1618 (2004).
  40. Mohammadi, A., Mehrzad, J., Mahmoudi, M., Schneider, M., Haghparast, A. Effect of culture and maturation on human monocyte-derived dendritic cell surface markers, necrosis and antigen binding. Biotech Histochem. 90, 445-452 (2015).

This article has been published

Video Coming Soon

JoVE Logo


Terms of Use





Copyright © 2024 MyJoVE Corporation. All rights reserved