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Cell Biol."],"published-print":{"date-parts":[[2026,1,1]]},"abstract":"<jats:p>Microglia, the primary immune cells of the brain parenchyma, play critical roles in neurodevelopment and homeostasis. The human microglia clone 3 (HMC3) cell line has long been a staple in vitro tool for exploring microglia dynamics. However, recent evidence suggests that HMC3 cells behave less like natural microglia and more like pericytes and astrocytes. Until a large-scale shift in replacing HMC3 cells with alternative in vitro models occurs, it is important to standardize characterization techniques used to study HMC3 polarization dynamics to enhance repeatability of findings. Here, we characterized the morphological, cellular, and molecular responses of HMC3 cells in response to 10 and 50\u2009ng\/mL interferon-gamma (IFN-\u03b3) treatment. We created an HMC3-specific morphology atlas, conducted manual and automated morphology assessments, quantified live and dead cell counts, and assessed mitochondrial output and the accumulation of reactive oxygen species. We also quantified transcript and protein abundance of candidate markers of chemokine secretion, cytokine signaling, endosomal processes, and cytoprotective responses. We presented detailed methodology on cell culturing, treatments, target selection, internal reference controls, primer design, validation, testing, and quantification to enhance the reproducibility of the data. 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