Monocyte Subsets by Flow Cytometry and Dendritic Cell Flow Cytometry Panel

Gating Strategy | Staining Protocol | Additional Flow Cytometry Resources

Immunophenotyping of Monocyte Cell and Dendritic Cell Subsets

Monocytes and dendritic cells of the innate immune system are critical mediators of the early host immune response. Use this validated flow cytometry panel to distinguish classical monocytes, non-classical monocytes, plasmacytoid dendritic cells (pDCs) and monocyte-derived dendritic cells (mDCs). Use this validated flow cytometry panel to identify classical monocytes, non-classical monocytes, pDCs and mDCs.

Flow Cytometry Panel for Immunophenotyping of Monocyte Cells and Dendritic Cells

MarkerCloneFluorochromeCatalog #
HLA-DRL203Alexa Fluor®405FAB4869V
CD8637301FITCFAB141F
CD11cICRF 3.9PEFAB1777P
CD12332703PerCPFAB301C
CD16245536APCFAB2546A
CD20396444Alexa Fluor®700FAB4225N
CD3UCHT1*Alexa Fluor®700FAB100N
CD562524CAlexa Fluor®700FAB24086N
CD14134620Alexa Fluor®750FAB3832S

*Designate clones independently validated by HLDA.
Alexa Fluor® is registered trademark of Molecular Probes, Inc.

This multicolor flow cytometry panel was validated on human peripheral blood mononuclear cells (PBMCs).

Flow Cytometry Gating Strategy for Monocyte Cell and Dendritic Cell Subsets Panel

Pseudocolor flow cytometry plot showing gating strategy for monocyte and dendritic cell subsets.

Multicolor flow cytometry panel to identify monocyte and dendritic cell subsets. Monocyte and dendritic cell subsets were determined first based on expression of CD14 and HLA-DR. CD14+ monocytes were then gated into CD16- Classical Monocytes and CD16+ Non-Classical Monocytes. CD14-HLA-DR+ Dendritic Cells were then gated into CD11c+ CD123- monocyte-derived dendritic cells (mDCs) and CD11c-CD123+ plasmacytoid dendritic cells (pDCs). Cells were stained with Anti-Human HLA-DR Alexa Fluor® 405, CD8 FITC, CD11c PE, CD123 PerCP, CD16 APC, CD20 Alexa Fluor® 700, CD3 Alexa Fluor® 700, CD56 Alexa Fluor® 700, and CD14 Alexa Fluor® 750.

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Staining Protocol for Monocyte Cell Subset Panel

Other Supplies Required

  1. Wash human PBMCs (1 x 106 cells per sample) with 2 mL of Staining Buffer (1X) (Catalog # FC001) or other BSA-containing buffer, by spinning at 300 x g for 5 minutes, using 5 mL flow cytometry tubes. Decant/aspirate supernatant.
  2. Fc-block cells with blocking IgG (1 μg IgG/106 cells) for 10 minutes at room temperature.
  3. Add previously titrated amount of each of the fluorochrome conjugated antibodies. Vortex tubes.

Monocyte Cell Antibody Concentration Chart  

  1. MarkerFluorochromeRecommended Concentration
    HLA-DRAlexa Fluor® 4055 μL/106 cells
    CD86FITC10 μL/106 cells
    CD11cPE10 μL/106 cells
    CD123PerCP10 μL/106 cells
    CD16APC10 μL/106 cells
    CD20Alexa Fluor® 7005 μL/106 cells
    CD3Alexa Fluor® 7005 μL/106 cells
    CD56Alexa Fluor® 7000.25-1 μg/106 cells
    CD14Alexa Fluor® 7505 μL/106 cells
  2. (Optional) To a separate tube, add 5 μL of each of the isotype control antibodies. Vortex tubes.
  3. Incubate the mixtures for 30-45 minutes at room temperature in the dark.
  4. At the end of the incubation, wash with 2 mL of Staining Buffer (1X), by spinning at 300 x g for 5 minutes. Decant/aspirate supernatant.
  5. Resuspend the cells in 0.2-0.5 mL Staining Buffer (1X) and acquire on a Flow Cytometer.
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Additional Flow Cytometry Products and Resources

Products:

Isotype Controls
MagCellect™ Cell Selection Kits
Quality Control and Standardization Beads from Novus Biologicals

Resources:

Flow Cytometry Handbook
Intracellular Staining with Alcohol Permeabilization Protocol
Intracellular Staining with Detergent Permeabilization Protocol
On-Demand Webinar: Demystifying Multi-parameter Flow Cytometry
On Demand Webinar: Turning Flow Cytometry Upside Down and Inside Out

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