Method for the simultaneous isolation of primary astrocytes and microglia from the neonatal rats cerebral cortex

Objective: To establish a protocol for the simultaneous isolation and high-purity purification of primary astrocytes and microglia from neonatal rats cerebral cortex. Methods: Single-cell suspensions were prepared from cerebral cortices of postnatal day 2 (P2) rat pups. Fibroblasts were pre-removed using differential adhesion techniques. Mixed glial cultures were maintained with graded serum (from 10% to 5% to 2% FBS) to suppress fibroblast proliferation. On day 14, microglia were isolated by constant temperature shaking (200 rpm, 12 h, 37 °C), followed by manual agitation of remaining adherent cells to purify astrocytes. Cell purity was assessed by immunofluorescence (Iba1 and GFAP) and validated by multicolor flow cytometry (CD11b/CD45; ACSA-2) and ER-TR7 fibroblast exclusion staining. Cell viability was evaluated by trypan blue exclusion and CCK-8 assay. Microglial morphology was quantified by cell body area, circularity index, and primary process number. Results: ; circularity 0.438 ± 0.095; processes 4.3 ± 0.8 per cell). Cell viability remained above 92% following key procedural steps and recovered to over 95% post-purification; CCK-8 assay confirmed full metabolic recovery. Conclusion: This study establishes a combined method utilizing graded serum and constant temperature shaking for glial cell isolation, enabling simultaneous acquisition of both major glial cell types from a single animal. This cost-effective protocol provides a practical tool for functional studies of neuroglial cells.