Probing and painting to get the full picture of macrophage polarisation

Macrophages play central roles in tissue repair and remodelling, host defence and immunopathology. Their ability to rapidly reprogram key functions in response to local cues makes them attractive targets for drug discovery. While this plasticity creates therapeutic opportunities, it also complicates high-throughput screening since multiple, transient functional states can coexist. To address this challenge, we developed a 384-well, single-cell pipeline that uses two well-established biomarkers, inducible nitric oxide synthase and Arginase 1, as a compact primary readout for macrophage state. We benchmarked this targeted approach against untargeted image-based profiling, Cell Painting and ChromaLIVE. In a pilot screen, the biomarker panel rapidly identified distinct, biologically relevant hit classes. The paint approaches provided complementary, high-dimensional information useful for resolving ambiguous calls, though at increased computational cost. A probe-first, paint-second workflow optimally balances throughput, biological interpretability and computational efficiency, enabling scalable discovery of macrophage modulators.