Quick summary (TL;DR)

Different labs use slightly different PMA and polarization timings; two robust options are (A) PMA 100 ng/mL for 48 h → rest 48 h, or (B) low-PMA 5–15 ng/mL for 24–72 h → rest 24 h. Both yield adherent macrophage-like THP-1 (M0). Use the rest period to reduce residual PMA effects.

Polarize to M1 with LPS (100 ng/mL) + IFN-γ (20 ng/mL) for 24–48–72 h (many groups use 24–48 h; 48–72 h gives stronger transcriptional responses).

Polarize to M2 with IL-4 (20 ng/mL) ± IL-13 (20 ng/mL) for 48–72 h.

Validate with a panel (surface markers + genes + secreted cytokines) rather than a single marker; include functional assays (cytokine ELISA, phagocytosis) and viability checks.

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Protocols (two reliable, published approaches)

A. “Standard / High-PMA” (widely used, Baxter et al. J Immunol Methods 2020)

1. Seed THP-1 at ~0.5–1×10^6 cells/mL in tissue-culture plate.

2. Add PMA 100 ng/mL, incubate 48 h.

3. Remove PMA medium, replace with PMA-free medium and rest 48 h (to reduce activation artefacts). At this point you have M0/differentiated macrophages.

B. “Low-PMA” (reduces PMA cytotoxicity/artifacts; use if you see cell death or altered responses)

1. Seed as above. Add PMA 5–15 ng/mL for 24–72 h (many labs use 5 or 15 ng/mL × 24 h then rest).

2. Wash, rest 24–48 h in PMA-free medium. Check CD14 upregulation/adhesion.

Polarization (apply after rest period):

M1 (classical): add LPS 100 ng/mL + IFN-γ 20 ng/mL for 24–48 h (or 48–72 h for stronger gene induction). Measure TNF-α, IL-6, iNOS (gene), CD80/CD86 (surface).

M2 (alternative): add IL-4 20 ng/mL ± IL-13 20 ng/mL for 48–72 h. Measure CD206 (MRC1), CD163, CCL17, CD200R, Arg1 (note human Arg1 can be variable).

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Recommended marker panels (use multiple readouts)

Surface markers (flow cytometry):

M1: CD80, CD86, HLA-DR / MHC II (upregulated).

M2: CD206 (MRC1), CD163, CD200R.

Also include pan-macrophage markers (CD11b, CD14) to confirm differentiation.

mRNA / gene markers (qPCR):

M1: TNF, IL1B, IL6, NOS2 (note: human NOS2/iNOS expression is less consistent than murine).

M2: MRC1 (CD206), CCL17, CCL22, CD163, ARG1 (use cautiously in human cells).

Secreted proteins (ELISA):

M1: TNF-α, IL-6, IL-12 (p70).

M2: IL-10, TGF-β (but IL-10 can come from many cells—interpret in context).

Functional checks:

Phagocytosis assay (FITC-dextran or fluorescent beads) — M1 and M2 differ in phagocytic behavior in some assays.

Nitric oxide (human macrophages: limited NO production — don’t rely on NO alone).

Metabolic readouts: glycolysis vs oxidative phosphorylation (Seahorse) where possible — M1 tends to be more glycolytic.

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Controls and best practices

Always include unstimulated M0 (PMA-differentiated but not polarized) as the baseline control.

Time course: check markers at 24, 48, and 72 h post-stimulation — some markers take longer to appear (e.g., CD206 increases over 48–72 h).

Verify viability (Trypan Blue or Annexin V) — high PMA or prolonged LPS can kill cells and skew readouts.

Batch effects: cytokine lots, LPS source/serotype, and PMA stock age can change responses — use the same lot for key reagents where possible.

Primary MDMs as gold standard: if possible compare to monocyte-derived macrophages from human blood (donor MDMs) to confirm polarisation phenotype. Many studies show similar trends but not identical profiles between THP-1 and primary MDMs.

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Troubleshooting / pitfalls

Residual PMA signalling: insufficient rest after PMA leads to a “pre-activated” phenotype. Always include a rest period (24–48 h) before polarization.

Over-reliance on single markers: e.g., ARG1 or iNOS alone is unreliable in human macrophages — use a panel (surface + cytokine + gene).

PMA concentration tradeoff: high PMA gives strong adherence but may change baseline gene expression; low PMA reduces artefacts but sometimes yields less robust adherence — pick based on your downstream assays and run a small optimization.

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Example, compact workflow you can paste into Methods

1. Seed THP-1 at 0.5×10^6 cells/mL in 6-well plates.

2. Differentiate with PMA 100 ng/mL for 48 h. Wash and rest in PMA-free RPMI for 48 h (M0).

3. M1 polarization: LPS 100 ng/mL + IFN-γ 20 ng/mL for 24–48 h.

4. M2 polarization: IL-4 20 ng/mL ± IL-13 20 ng/mL for 48–72 h.

5. Readouts: flow (CD80, CD86, CD206, CD163, CD11b), qPCR (TNF, IL1B, IL6, MRC1, CCL17), ELISA (TNF-α, IL-6, IL-10), phagocytosis assay, viability.

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Useful primary references / protocol sources (for your methods & citation)

1. Baxter EW et al., Standardized protocols for differentiation of THP-1 cells to macrophages with distinct M(IFNγ+LPS), M(IL-4) and M(IL-10) phenotypes. J Immunol Methods. 2020.

2. Daigneault M. et al., The phorbol 12-myristate-13-acetate differentiation protocol is... (discussion of PMA dose/time effects). PMC article.

3. Chanput W. et al., M1 and M2 macrophages derived from THP-1 cells differentially … (polarization conditions e.g., IL-4/IL-13 20 ng/mL × 72 h). PMC article.

4. Szulc-Bray A. et al. / comparative THP-1 vs MDM studies (on activation and functional readouts). PMC reviews.

5. Recent reviews on macrophage markers and plasticity (for interpretation of markers like CD163, CD206, CD80/86).