A broadly adaptable protocol for isolating Kupffer cells from non-model species: application to

Background: Kupffer cells are specialized, liver-resident macrophages that play central roles in hepatic homeostasis, immune surveillance and pathogen clearance. While well-studied in laboratory mice and rats, methods for isolating and studying Kupffer cells from non-model species remain scarce, limiting research in ecologically and zoonotically relevant hosts such as the Natal multimammate mouse ( Methodology and principal findings: We developed and validated an optimized Kupffer cell isolation protocol adaptable to non-model rodents, relying on mechanical and enzymatic liver dissociation, non-parenchymal cell enrichment by Percoll gradient and selection by adherence. Critical parameters affecting yield and viability included maintaining all perfusion and digestion steps at 37 °C, limiting enzymatic digestion to ≤15min and avoiding aggressive mechanical disruption. Under optimized conditions, yields averaged 2.55 ± 1.13 × 10 Conclusions and significance: This protocol provides a robust, reproducible, and species-flexible method for isolating viable Kupffer cells from non-model rodents without requiring species-specific reagents. It enables functional, phenotypic, and virological studies in primary hepatic macrophages from