PMID- 27805582
OWN - NLM
STAT- MEDLINE
DCOM- 20170803
LR  - 20220330
IS  - 1940-087X (Electronic)
IS  - 1940-087X (Linking)
IP  - 116
DP  - 2016 Oct 18
TI  - Characterization of Human Monocyte-derived Dendritic Cells by Imaging Flow 
      Cytometry: A Comparison between Two Monocyte Isolation Protocols.
LID - 10.3791/54296 [doi]
LID - 54296
AB  - 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.
FAU - Figueroa, Gloria
AU  - Figueroa G
AD  - Department of Immunology, Herbert Wertheim College of Medicine, Florida 
      International University.
FAU - Parira, Tiyash
AU  - Parira T
AD  - Department of Immunology, Herbert Wertheim College of Medicine, Florida 
      International University.
FAU - Laverde, Alejandra
AU  - Laverde A
AD  - Department of Immunology, Herbert Wertheim College of Medicine, Florida 
      International University.
FAU - Casteleiro, Gianna
AU  - Casteleiro G
AD  - Department of Immunology, Herbert Wertheim College of Medicine, Florida 
      International University.
FAU - El-Mabhouh, Amal
AU  - El-Mabhouh A
AD  - Millipore Sigma.
FAU - Nair, Madhavan
AU  - Nair M
AD  - Department of Immunology, Herbert Wertheim College of Medicine, Florida 
      International University.
FAU - Agudelo, Marisela
AU  - Agudelo M
AD  - Department of Immunology, Herbert Wertheim College of Medicine, Florida 
      International University; magudelo@fiu.edu.
LA  - eng
GR  - K99 AA021264/AA/NIAAA NIH HHS/United States
GR  - R00 AA021264/AA/NIAAA NIH HHS/United States
GR  - R25 GM061347/GM/NIGMS NIH HHS/United States
PT  - Journal Article
PT  - Video-Audio Media
DEP - 20161018
PL  - United States
TA  - J Vis Exp
JT  - Journal of visualized experiments : JoVE
JID - 101313252
SB  - IM
MH  - Cell Differentiation
MH  - Cell Separation
MH  - Cells, Cultured
MH  - *Dendritic Cells
MH  - *Flow Cytometry
MH  - Humans
MH  - *Leukocytes, Mononuclear
MH  - Monocytes
PMC - PMC5092205
EDAT- 2016/11/03 06:00
MHDA- 2017/08/05 06:00
PMCR- 2018/10/18
CRDT- 2016/11/03 06:00
PHST- 2016/11/03 06:00 [pubmed]
PHST- 2017/08/05 06:00 [medline]
PHST- 2016/11/03 06:00 [entrez]
PHST- 2018/10/18 00:00 [pmc-release]
AID - 54296 [pii]
AID - 10.3791/54296 [doi]
PST - epublish
SO  - J Vis Exp. 2016 Oct 18;(116):54296. doi: 10.3791/54296.