Background: Macrophages (Mφ) are innate immune cells known for their different activation phenotypes, classically described as falling within two broad categories, M1 and M2. The latter were originally described as alternatively activated M2 cells to differentiate them from classically activated M1 cells. M2 cells were later classified into M2a (interleukin (IL)-4), M2b (immune complex), M2c (IL-10) and M2d (5-(N-ethylcarboxamido) adenosine (NECA) + lipopolysaccharide (LPS)) based on their inducing stimuli. Considering the established role of M2d/tumour-associated macrophage (TAM) cells within cancer initiation and proliferation, expanding on the knowledge of M2d characteristics can provide fundamental information for Mφ targeted immunotherapy. The precise characterization of M2d cells derived from tissues has not been described in detail., Methods: Our study focused on spleen-derived macrophages (SpM), which were also compared to bone marrow-derived macrophages (BMDMs)., Results: By investigating different conditions for M2d-specific stimulation and employing various assays including functional tests, we show how Mφ M2d (NECA + LPS) polarization can be affected by prolonged culture conditions to induce a phenotype that was clearly different from M2a cells., Conclusion: This work offers new insights into the properties of primary M2d Mφ following extended stimulation with LPS and NECA., (© 2025 The Author(s). Published by IMR Press.)