M1 Macrophage Flow Cytometry Panel

Immunophenotyping of M1 Macrophages

M1 Macrophages are associated with a high level of phagocytosis and secretion of pro-inflammatory cytokines. Their function is critical to host anti-tumor defense against cancer. Use this validated flow cytometry panel to identify and phenotype M1 Macrophages.

 

 

Flow Cytometry Antibodies for Immunophenotyping of M1 Macrophages

Marker Clone Fluorochrome Catalog #
CD38 240742 APC FAB2404A
CD11c ICRF 3.9 PE FAB1777P
HLA-DR L203 PerCP FAB4869C
CD86 37301 FITC FAB141F
NCAM1/CD56 2524C Alexa Fluor®700 FAB24086N
CD20 396444 Alexa Fluor®405 FAB4225V
CD3 UCHT1* Alexa Fluor®405 FAB100V

*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 M1 Macrophage Polarization Panel

Pseudocolor flow cytometry plot showing gating strategy for NK Cell markers CD56, CD38, CD11c, HLA-DR, CD86, CD20, and CD3.

Multicolor flow cytometry panel to identify M1 Macrophages. Monocytes were isolated from PBMCs via adherence depletion for 5 hours. Non-adherent cells were removed. Adherent cells were incubated for 6 days in C3 +10% huAB media with 50 ng/ml Recombinant Human GM-CSF (Catalog # 215-GM), media and cytokines are refreshed on day 3. For M1 polarization, 24 hours prior to staining, cells are incubated with 50 ng/ml human GM-CSF (Catalog # 215-GM ), 50 ng/ml Recombinant Human IFN-gamma (Catalog # 285-IF), and 50 ng/ml LPS. Cells were stained with Anti-human CD3 Alexa Fluor® 405, CD20 Alexa Fluor® 405, CD56 Alexa Fluor® 700, CD86 FITC, HLA-DR PerCP, CD11c PE, and CD38 APC. Cells were previously gated on Live CD3-CD20-CD56-.

 

 

Staining Protocol for M1 Macrophage Polarization 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 primary conjugated antibody. Vortex tubes.

    M1 Flow Cytometry Antibody Concentration Chart  
    Marker Fluorochrome Recommended Concentration
    CD38 APC 10 μL/106 cells
    CD11c PE 10 μL/106 cells
    HLA-DR PerCP 10 μL/106 cells
    CD86 FITC 10 μL/106 cells
    NCAM1/CD56 Alexa Fluor® 700 0.25-1 μg/106 cells
    CD20 Alexa Fluor® 405 5 μL/106 cells
    CD3 Alexa Fluor® 405 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.