Protocol for Simultaneous Isolation of Primary Astrocytes and Microglia from Neonatal Rats

Supplementary file 1_Method for the simultaneous isolation of primary astrocytes and microglia from the neonatal rats cerebral cortex.docx is a 2.9 MB document by Kun Zhang, last updated April 21, 2026. It details a laboratory protocol for obtaining high-purity primary glial cells from postnatal day 2 rat pups. The method uses differential adhesion and graded serum to suppress fibroblasts, followed by constant temperature shaking to separate microglia and astrocytes.

Use Cases

• Replicating a high-purity glial cell isolation protocol based on the detailed methodological steps.
• Comparing cell purity and viability metrics from different isolation techniques based on the reported immunofluorescence and flow cytometry results.
• Studying microglial morphological development over time based on the quantified cell body area, circularity, and process number data.
• Validating fibroblast exclusion methods in primary cell culture based on the ER-TR7 staining results described.

Strengths

• Reports high cell purity, with microglia at 98.98% ± 1.21% and astrocytes at 98.81% ± 2.38% after purification.
• Includes specific quantitative metrics for microglial morphology, such as cell body area transitioning from 173.5 ± 32.8 μm² to 418.2 ± 68.3 μm².
• Validates results using multiple techniques: immunofluorescence (Iba1, GFAP), multicolor flow cytometry (CD11b/CD45, ACSA-2), and fibroblast exclusion staining (ER-TR7).
• Documents cell viability remaining above 92% during procedures and recovering to over 95% post-purification.

Limitations

• Column-level documentation is absent; field semantics must be inferred after download.
• Row count is unknown, which may limit suitability assessment.
• Data is specific to postnatal day 2 Sprague-Dawley rat pups, which may limit generalizability to other species or developmental stages.

Provenance

Source

Kun Zhang via figshare.

Collection Method

Laboratory protocol involving tissue dissociation, differential adhesion, graded serum culture, and mechanical shaking.

Time Range

Experimental data likely corresponds to the study period, but specific range is not stated.

Freshness

Last updated 2026-04-21 04:22:16; freshness should be verified.