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.

The NK Cell Phenotype Panel is also available as a multicolor flow cytometry kit (Catalog # FMC033) complete with isotype controls and staining buffer.

 

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.

Flow cytometry analysis of 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. Live CD3-CD20-CD56- cells were analyzed for expression of CD38, CD11c, HLA-DR, and CD86.

 

 

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 5 μL (or previously titrated amount) of each primary conjugated antibody. 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.