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

*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
 

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

• PBS
• Flow Cytometry Staining Buffer (Catalog # FC001)
• Fc-block (blocking IgG)
• (Optional) Isotype Control Antibodies
• 5 mL Flow cytometry tubes

Protocol Overview

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.
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.

M1 Flow Cytometry Antibody Concentration Chart

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