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

Day 14 was identified as the optimal separation time point. Immediately post-shaking, microglia purity (Iba1+) reached 98.6% ± 1.1%, and astrocyte purity (GFAP+) was 98.4% ± 1.7%. After subsequent purification culture, these values increased to 98.98% ± 1.21% and 98.81% ± 2.38%, respectively. Dual-label immunofluorescence confirmed minimal cross-contamination, with Iba1+/GFAP+ dual-positive cells constituting <1% in both populations. Multicolor flow cytometry corroborated these findings, yielding CD11b+ purity of 97.12% ± 1.58% for microglia (with 95.37% ± 1.84% classified as CD11b+/CD45^low homeostatic microglia) and ACSA-2+ purity of 94.65% ± 2.73% for astrocytes. No unequivocal ER-TR7+ fibroblasts were identified in either purified population. Microglial morphology progressively transitioned from amoeboid (Day 0: area 173.5 ± 32.8 μm2; circularity 0.847 ± 0.058; processes 0.8 ± 0.4) to ramified (Day 5: area 418.2 ± 68.3 μm2; 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.