A Multidimensional Optimization Strategy for High-Purity Primary Rat Microglia Isolation with Preserved Functional Responsiveness.

BACKGROUND: Primary microglia are essential for studying neuroinflammation and microglia-mediated neuropathology. However, conventional shaking-based isolation methods often yield unstable purity, astrocytic contamination, and heterogeneous activation states. NEW METHOD: We developed a multidimensional optimization strategy for primary rat microglia isolation by systematically integrating three key parameters: neonatal developmental stage, culture vessel geometry, and Percoll density gradient purification. Microglial purity, identity, viability, and functional responsiveness were evaluated by flow cytometry, immunofluorescence, Western blotting, qPCR, and ELISA. RESULTS: Compared with postnatal day 7 (P7), postnatal day 3 (P3) tissue provided higher isolation efficiency, greater culture homogeneity, and reduced astrocytic contamination. Culture in 6-cm dishes improved cell adhesion and morphological consistency. Percoll density gradient purification further increased microglial purity by approximately 20-30% while maintaining acceptable cell recovery. The optimized protocol consistently yielded cultures with stable purity (80-90%), high IBA1 positivity (>90%), increased metabolic activity, and lower basal activation. Following lipopolysaccharide stimulation, purified microglia exhibited robust inflammatory responses, including increased cytokine secretion and inflammatory gene expression. COMPARISON WITH EXISTING METHODS: Compared with conventional shaking-based isolation, the optimized workflow improves purity, reduces contamination, enhances reproducibility, and preserves functional responsiveness without requiring specialized equipment. CONCLUSIONS: This study provides a practical and reproducible strategy for improving microglial purity and experimental consistency and offers a reliable experimental platform for neuroinflammation research and mechanistic studies.