Monocyte and Dendritic Cell Subsets Flow Cytometry Panel

Immunophenotyping of Monocyte 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).

 

 

Flow Cytometry Panel for Immunophenotyping of Monocytes 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.

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

 

Flow Cytometry Gating Strategy for Monocyte and Dendritic Cell Subsets Panel

Pseudocolor flow cytometry plot showing gating strategy for M2 Macrophage polarization showing expression of VEGF, CD204, CD163, and CD206.

Flow cytometry analysis of monocyte and dendritic cell subsets in PBMCs. 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).

 

 

Staining Protocol for Monocyte 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 5 μL (or previously titrated amount) of each surface marker (CD3 Alexa Fluor® 405, CD20 Alexa Fluor® 405, and CD56 Alexa Fluor® 700). Vortex tubes.
  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.