{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,4,21]],"date-time":"2026-04-21T04:46:28Z","timestamp":1776746788840,"version":"3.51.2"},"reference-count":53,"publisher":"Frontiers Media SA","license":[{"start":{"date-parts":[[2026,4,21]],"date-time":"2026-04-21T00:00:00Z","timestamp":1776729600000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"content-domain":{"domain":["frontiersin.org"],"crossmark-restriction":true},"short-container-title":["Front. Cell. Neurosci."],"abstract":"<jats:sec>\n                    <jats:title>Objective<\/jats:title>\n                    <jats:p>To establish a protocol for the simultaneous isolation and high-purity purification of primary astrocytes and microglia from neonatal rats cerebral cortex.<\/jats:p>\n                  <\/jats:sec>\n                  <jats:sec>\n                    <jats:title>Methods<\/jats:title>\n                    <jats:p>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 \u00b0C), 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.<\/jats:p>\n                  <\/jats:sec>\n                  <jats:sec>\n                    <jats:title>Results<\/jats:title>\n                    <jats:p>\n                      Day 14 was identified as the optimal separation time point. Immediately post-shaking, microglia purity (Iba1\n                      <jats:sup>+<\/jats:sup>\n                      ) reached 98.6% \u00b1 1.1%, and astrocyte purity (GFAP\n                      <jats:sup>+<\/jats:sup>\n                      ) was 98.4% \u00b1 1.7%. After subsequent purification culture, these values increased to 98.98% \u00b1 1.21% and 98.81% \u00b1 2.38%, respectively. Dual-label immunofluorescence confirmed minimal cross-contamination, with Iba1\n                      <jats:sup>+<\/jats:sup>\n                      \/GFAP\n                      <jats:sup>+<\/jats:sup>\n                      dual-positive cells constituting &amp;lt;1% in both populations. Multicolor flow cytometry corroborated these findings, yielding CD11b\n                      <jats:sup>+<\/jats:sup>\n                      purity of 97.12% \u00b1 1.58% for microglia (with 95.37% \u00b1 1.84% classified as CD11b\n                      <jats:sup>+<\/jats:sup>\n                      \/CD45^low homeostatic microglia) and ACSA-2\n                      <jats:sup>+<\/jats:sup>\n                      purity of 94.65% \u00b1 2.73% for astrocytes. No unequivocal ER-TR7\n                      <jats:sup>+<\/jats:sup>\n                      fibroblasts were identified in either purified population. Microglial morphology progressively transitioned from amoeboid (Day 0: area 173.5 \u00b1 32.8 \u03bcm\n                      <jats:sup>2<\/jats:sup>\n                      ; circularity 0.847 \u00b1 0.058; processes 0.8 \u00b1 0.4) to ramified (Day 5: area 418.2 \u00b1 68.3 \u03bcm\n                      <jats:sup>2<\/jats:sup>\n                      ; circularity 0.438 \u00b1 0.095; processes 4.3 \u00b1 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.\n                    <\/jats:p>\n                  <\/jats:sec>\n                  <jats:sec>\n                    <jats:title>Conclusion<\/jats:title>\n                    <jats:p>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.<\/jats:p>\n                  <\/jats:sec>","DOI":"10.3389\/fncel.2026.1787397","type":"journal-article","created":{"date-parts":[[2026,4,21]],"date-time":"2026-04-21T04:22:38Z","timestamp":1776745358000},"update-policy":"https:\/\/doi.org\/10.3389\/crossmark-policy","source":"Crossref","is-referenced-by-count":0,"title":["Method for the simultaneous isolation of primary astrocytes and microglia from the neonatal rats cerebral cortex"],"prefix":"10.3389","volume":"20","author":[{"given":"Kun","family":"Zhang","sequence":"first","affiliation":[{"name":"College of Clinical Medicine, Guizhou Medical University","place":["Guiyang, China"]}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Songcan","family":"Wu","sequence":"additional","affiliation":[{"name":"College of Clinical Medicine, Guizhou Medical University","place":["Guiyang, China"]}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Yujie","family":"Zeng","sequence":"additional","affiliation":[{"name":"College of Clinical Medicine, Guizhou Medical University","place":["Guiyang, China"]}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Xinglin","family":"Wu","sequence":"additional","affiliation":[{"name":"College of Clinical Medicine, Guizhou Medical University","place":["Guiyang, China"]}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Meiming","family":"Qian","sequence":"additional","affiliation":[{"name":"College of Clinical Medicine, Guizhou Medical University","place":["Guiyang, China"]}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Kaya","family":"Xu","sequence":"additional","affiliation":[{"name":"College of Clinical Medicine, Guizhou Medical University","place":["Guiyang, China"]},{"name":"Department of Neurosurgery, Affiliated Hospital of Guizhou Medical University","place":["Guiyang, China"]}],"role":[{"role":"author","vocabulary":"crossref"}]}],"member":"1965","published-online":{"date-parts":[[2026,4,21]]},"reference":[{"key":"B1","doi-asserted-by":"publisher","first-page":"396","DOI":"10.1016\/j.stem.2020.07.008","article-title":"Distinct regulatory programs control the latent regenerative potential of dermal fibroblasts during wound healing.","volume":"27","author":"Abbasi","year":"2020","journal-title":"Cell Stem Cell"},{"key":"B2","doi-asserted-by":"publisher","first-page":"108742","DOI":"10.1016\/j.jneumeth.2020.108742","article-title":"Isolation, culture, and downstream characterization of primary microglia and astrocytes from adult rodent brain and spinal cord.","volume":"340","author":"Agalave","year":"2020","journal-title":"J. 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