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M2 Macrophage Flow Cytometry Panel

Immunophenotyping of M2 Macrophage Polarization

Often referred to as tumor associated macrophages (TAMs), M2 macrophages are more likely to express inhibitory ligands and secrete anti-inflammatory cytokines. M2 macrophages can inhibit NK Cell cytotoxicity by expressing non-classical MHC I molecules. Use this validated flow cytometry panel to phenotype M2 Macrophages.

 

Gating Strategy

Staining Protocol

Additional Flow Cytometry Resources

 

Flow Cytometry Antibodies for Immunophenotyping of M2 Macrophages

Marker Clone Fluorochrome Catalog #
VEGF 23410 APC IC2931A
SR-AI/MSR/CD204 351615 PE FAB2708P
CD163 215927 PerCP FAB1607C
MMR/CD206 685641 Alexa Fluor®488 FAB25342G
NCAM-1/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 M2 Macrophage Polarization Panel

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

Multicolor flow cytometry panel to identify M2 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 M-CSF (Catalog # 216-MC), media and cytokines are refreshed on day 3. For M2 polarization, 24 hours prior to staining, cells are incubated with 50 ng/ml human M-CSF (Catalog # 216-MC), 20 ng/ml Recombinant Human IL-4 (Catalog # 204-IL), and 20 ng/ml Recombinant Human IL-13 (Catalog # 213-ILB). Cells were stained with Anti-Human CD3 Alexa Fluor® 405, CD20 Alexa Fluor® 405, CD56 Alexa Fluor® 700, CD206 Alexa Fluor® 488, CD163 PerCP, CD204 PE, and VEGF APC. Cells were previously gated on Live CD3-CD20-CD56-.

 

Staining Protocol for M2 Macrophage Polarization Panel

Other Supplies Required

Surface Stain

  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 surface marker. Vortex tubes.

     M2 Flow Cytometry Antibody Concentration Chart
    Marker Fluorochrome Recommended Concentration
    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
    CD163 PerCP 10 μL/106 cells
    MMR/CD206 Alexa Fluor® 488 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.

Intracellular Stain (with alcohol permeabilization)

  1. Add 0.5 mL of cold Flow Cytometry Fixation Buffer (Catalog # FC004) and vortex. Incubate at room temperature for 10 minutes. Vortex cells intermittently in order to maintain a single cell suspension.
  2. Centrifuge cells 300-500 x g for 5 minutes. Decant the Fixation Buffer.
  3. Wash cells PBS (or HBSS) by adding 2 mL of PBS (or HBSS), centrifuge at 350-500 x g for 5 minutes, and decant buffer from pelleted cells. Repeat (2 total washes).
  4. Resuspend cells in 900 μL of -20 °C methanol. Incubate for 30 minutes at 4 °C.
  5. Centrifuge cells for 5 minutes at 350-500 x g. Remove and discard the supernatant. Wash 2 times with PBS (or HBSS) as described in step 1.
  6. Add previously titrated amount of conjugated antibody to intracellular activation markers. Vortex tubes.

    M2 Flow Cytometry Antibody Concentration Chart  
    Marker Fluorochrome Recommended Concentration
    VEGF APC 10 μL/106 cells
    SR-AI/MSR/CD204 PE 10 μL/106 cells
     
  7. Wash cells 2 times with PBS (or HBSS) as described in step 3.
    Note: If an unconjugated primary antibody is used, incubation with an appropriate secondary antibody should occur now. Dilute the secondary antibody in PBS (or HBSS), starting with the concentration suggested in the product datasheet. Incubate for 20-30 minutes in the dark and wash as in step 3.
  8. Resuspend cell pellet in 200 - 400 μL of PBS for flow cytometric analysis.

 

 

Additional Flow Cytometry Products and Resources

Products:

Isotype Controls
MagCellect™ Cell Selection Kits
Quality Control and Standardization Beads from Novus Biologicals

Resources:

Flow Cytometry Handbook
Intracellular Staining with Alcohol Permeabilization Protocol
Intracellular Staining with Detergent Permeabilization Protocol
On-Demand Webinar: Demystifying Multi-parameter Flow Cytometry
On Demand Webinar: Turning Flow Cytometry Upside Down and Inside Out