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.

 

 

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.

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.

Flow cytometry analysis of 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). Live CD3-CD20-CD56- cells were analyzed for expression of VEGF, CD204, CD163 ,and CD206.

 

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 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.

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 5-10 µL/106 cells (or a previously titrated amount)of conjugated antibody to intracellular activation markers (CD206 Alexa Fluor® 488, CD163 PerCP, VEGF APC, and CD204 PE). Vortex tubes.
  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.