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(1) The monocytic leukemia cell line THP-1 and primary monocyte-derived macrophages (MDMs) are popular in vitro model systems to study human innate immunity, wound healing, and tissue regeneration. However, both cell types differ significantly in their origin and response to activation stimuli. (2) Resting THP-1 and MDMs were stimulated with lipopolysaccharide (LPS) and interferon γ (IFNγ) and analyzed by Raman microspectroscopy (RM) before and 48 h after activation. Raman data were subsequently analyzed using principal component analysis. (3) We were able to resolve and analyze the spatial distribution and molecular composition of proteins, nucleic acids, and lipids in resting and activated THP-1 and MDMs. Our findings reveal that proinflammatory activation-induced significant spectral alterations at protein and phospholipid levels in THP-1. In MDMs, we identified that nucleic acid and non-membrane-associated intracellular lipid composition were also affected. (4) Our results show that it is crucial to carefully choose the right cell type for an in vitro model as the nature of the cells itself may impact immune cell polarization or activation results. Moreover, we demonstrated that RM is a sensitive tool for investigating cell-specific responses to activation stimuli and monitoring molecular changes in subcellular structures.

Raman Microspectroscopy Identifies Biochemical Activation Fingerprints in THP-1- and PBMC-Derived Macrophages