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Updated: Aug 9, 2026
Characterization of Human Monocyte-derived Dendritic Cells by Imaging Flow Cytometry: A Comparison between Two Monocyte Isolation Protocols
Published on: October 18, 2016
An alternative flow cytometry strategy for peripheral blood dendritic cell enumeration in the setting of repetitive
Kehui Wang1, Kevin P Nishimoto, Rita S Mehta
1Department of Medicine, Division of Hematology/Oncology, School of Medicine, University of California, Irvine, USA. kwang@uci.edu
Insights
A new antibody cocktail using CD66 instead of CD14 improves the identification of circulating dendritic cells (DCs). This method offers more accurate and reliable enumeration of DCs, especially during granulocyte macrophage colony-stimulating factor (GM-CSF) therapy.
Area of Science:
- Immunology
- Cell Biology
- Flow Cytometry
Background:
- Enumerating circulating peripheral blood dendritic cells (DCs) is challenging due to the lack of a unique surface marker and the use of adjuvants like granulocyte macrophage colony-stimulating factor (GM-CSF).
- Current methods often use a CD14-containing antibody cocktail to identify a lineage-negative, MHC class II-positive population, but this can be complicated by CD14 expression on some DC subsets and variable gating strategies.
- Increasing understanding of DC diversity and GM-CSF effects necessitates improved methods for accurate DC identification.
Purpose of the Study:
- To evaluate an alternative lineage antibody cocktail, replacing anti-CD14 with anti-CD66, for more accurate enumeration of circulating dendritic cells (DCs).
- To compare the efficacy of the standard CD14-based cocktail versus the alternative CD66-based cocktail in identifying DCs in normal donors and breast cancer patients undergoing chemotherapy with GM-CSF support.
Main Methods:
- Whole blood samples from normal donors and breast cancer patients were stained using fluorochrome-conjugated antibodies against MHC class II and either a standard CD14-containing or an alternative CD66acde-containing lineage cocktail.
- Putative DCs were enumerated via standard flow cytometry, with data analyzed using Mann-Whitney or Wilcoxon signed rank tests.
- Cellular morphology of sorted populations was examined post-GM-CSF treatment.
Main Results:
- Both antibody cocktails identified comparable lineage-negative, MHC class II-positive populations in normal donors and at baseline in cancer patients.
- The alternative CD66-based cocktail identified larger putative DC populations with increasing MHC class II expression and yielded more homogenous and consistent results post-GM-CSF.
- The CD66-based cocktail demonstrated less variation across gating strategies and more uniform longitudinal data, aligning with known GM-CSF activity.
Conclusions:
- An alternative lineage cocktail substituting anti-CD66 for anti-CD14 is a viable and potentially more accurate method for enumerating circulating DCs.
- This approach may improve the definition of the circulating DC pool by including CD14-positive immature DCs.
- The CD66-based cocktail offers more reliable data, particularly in contexts involving sustained GM-CSF administration.
Background:
Enumeration of circulating peripheral blood dendritic cells (DCs) is complicated by the absence of a unique cell surface marker expressed on all DC subsets and by the use of various biological adjuvants to modulate the DC compartment, including granulocyte macrophage colony stimulating factor (GM-CSF). Common methods employ a cocktail of antibodies, typically including anti-CD14, to define a lineage negative, MHC class II positive, putative DC population. Reported flow cytometry protocols include highly variable gating strategies and DC identification criteria. Increasing appreciation of DC pleiomorphism, GM-CSF biology, and recognition of CD14 expression in some DC subsets led us to consider an alternative lineage cocktail to improve identification of the circulating DC pool.
Methods:
Standard whole blood staining with appropriate fluorochrome conjugated antibodies to MHC class II and either standard CD14 containing, or an alternate CD66acde containing, lineage cocktail was performed on samples obtained from normal donors and breast cancer patients before and after administration of dose-dense, cytotoxic chemotherapy with daily GM-CSF hematopoetic growth factor support. Putative DCs were enumerated by standard flow cytometry. Data set differences were evaluated using two tailed Mann-Whitney or Wilcoxon signed rank tests. Cellular morphology was examined in cell-sorted populations from post GM-CSF samples.
Results:
Use of either antibody cocktail defined comparably sized lineage negative, MHC class II positive populations in normal donors and at baseline in cancer patients. However, selection of lineage negative subsets with increasing MHC class II expression levels yielded larger putative DC populations identified with the alternate cocktail. Both cocktails yielded highly reproducible data. Use of the alternate cocktail: 1) yielded a putative DC population, post GM-CSF that was more homogenous and consistent with DCs, 2) resulted in less data variation across gating strategies, and 3) resulted in more uniform and concordant longitudinal data, consistent with established GM-CSF biological activity.
Conclusion:
An alternative lineage negative cocktail substituting anti-CD66 antibody for anti-CD14 is a viable option for enumerating the circulating DC population, potentially more accurately defining the circulating DC pool by including CD14 positive immature DCs, and thus, may give more reliable data, particularly in the setting of sustained GM-CSF administration.
