Monocyte Subsets by Flow Cytometry and Dendritic Cell Flow Cytometry Panel

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

Marker Clone Fluorochrome Catalog #
HLA-DR L203 Alexa Fluor®405 FAB4869V
CD86 37301 FITC FAB141F
CD11c ICRF 3.9 PE FAB1777P
CD123 32703 PerCP FAB301C
CD16 245536 APC FAB2546A
CD20 396444 Alexa Fluor®700 FAB4225N
CD3 UCHT1* Alexa Fluor®700 FAB100N
CD56 2524C Alexa Fluor®700 FAB24086N
CD14 134620 Alexa Fluor®750 FAB3832S

*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 including CD16, CD14, CD11c, CD123, and HLA-DR.

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.



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  
    Marker Fluorochrome Recommended Concentration
    HLA-DR Alexa Fluor® 405 5 μL/106 cells
    CD86 FITC 10 μL/106 cells
    CD11c PE 10 μL/106 cells
    CD123 PerCP 10 μL/106 cells
    CD16 APC 10 μL/106 cells
    CD20 Alexa Fluor® 700 5 μL/106 cells
    CD3 Alexa Fluor® 700 5 μL/106 cells
    CD56 Alexa Fluor® 700 0.25-1 μg/106 cells
    CD14 Alexa Fluor® 750 5 μL/106 cells
  4. (Optional) To a separate tube, add 5 μL of each of the isotype control antibodies. Vortex tubes.
  5. Incubate the mixtures for 30-45 minutes at room temperature in the dark.
  6. 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.
  7. Resuspend the cells in 0.2-0.5 mL Staining Buffer (1X) and acquire on a Flow Cytometer.