Protocol for sex-specific isolation of microglia, oligodendrocyte precursor cells, and astrocytes from neonatal rats using shake-off technique

Gen Hamanaka; Tomonori Hoshino; Hidehiro Ishikawa; Akane Mizutani; Akihiro Shindo; Ken Arai

doi:10.1016/j.xpro.2025.103902

Protocol for sex-specific isolation of microglia, oligodendrocyte precursor cells, and astrocytes from neonatal rats using shake-off technique

STAR Protoc. 2025 Sep 19;6(3):103902. doi: 10.1016/j.xpro.2025.103902. Epub 2025 Jun 18.

Authors

Gen Hamanaka¹, Tomonori Hoshino², Hidehiro Ishikawa³, Akane Mizutani⁴, Akihiro Shindo⁴, Ken Arai⁵

Affiliations

¹ Neuroprotection Research Laboratories, Departments of Radiology and Neurology, Massachusetts General Hospital and Harvard Medical School, Charlestown, MA 02129-2000, USA. Electronic address: ghamanaka@mgh.harvard.edu.
² Neuroprotection Research Laboratories, Departments of Radiology and Neurology, Massachusetts General Hospital and Harvard Medical School, Charlestown, MA 02129-2000, USA.
³ Neuroprotection Research Laboratories, Departments of Radiology and Neurology, Massachusetts General Hospital and Harvard Medical School, Charlestown, MA 02129-2000, USA; Department of Neurology, Mie University Graduate School of Medicine, 2-174 Edobashi, Tsu, Mie 514-8507, Japan.
⁴ Department of Neurology, Mie University Graduate School of Medicine, 2-174 Edobashi, Tsu, Mie 514-8507, Japan.
⁵ Neuroprotection Research Laboratories, Departments of Radiology and Neurology, Massachusetts General Hospital and Harvard Medical School, Charlestown, MA 02129-2000, USA. Electronic address: karai@partners.org.

Abstract

Disease-related glial changes often show sex-specific differences, yet protocols for sex-specific isolation are limited. Here, we present a protocol for sex-specific isolation of microglia, oligodendrocyte precursor cells (OPCs), and astrocytes from neonatal rats using the shake-off technique. We describe steps for determining pup sex at post-natal days 1-2 and for dissecting, dissociating, and plating cortex cells. We then detail isolation procedures for each cell type. Downstream applications include immunocytochemistry, quantitative PCR, and RNA sequencing to assess cell-specific functions.

Keywords: Cell Differentiation; Cell culture; Cell isolation; Neuroscience.

MeSH terms

Animals
Animals, Newborn
Astrocytes* / cytology
Cell Separation* / methods
Female
Male
Microglia* / cytology
Oligodendrocyte Precursor Cells* / cytology
Rats
Sex Characteristics