ACSA2 is not astrocyte-specific: implications for cell sorting strategies in the rodent brain

Emerson Daniele; Gabriel Khelifi; Daniel Beretta; Boyan K Tsankov; K W Annie Bang; Chiara Beretta; Dana J Philpott; Samer M Hussein; Maryam Faiz

doi:10.3389/fncel.2026.1756677

ACSA2 is not astrocyte-specific: implications for cell sorting strategies in the rodent brain

Front Cell Neurosci. 2026 Feb 18:20:1756677. doi: 10.3389/fncel.2026.1756677. eCollection 2026.

Authors

Emerson Daniele¹, Gabriel Khelifi^{2

3

4}, Daniel Beretta⁵, Boyan K Tsankov⁶, K W Annie Bang⁷, Chiara Beretta^{5

8

9}, Dana J Philpott^{1

6

10}, Samer M Hussein^{2

3

4}, Maryam Faiz^{1

5

8

9

10

11}

Affiliations

¹ Institute of Medical Science, University of Toronto, Toronto, ON, Canada.
² Department of Molecular Biology, Medical Biochemistry and Pathology, Faculty of Medicine, Université Laval, Québec City, QC, Canada.
³ Oncology Division, CHU de Québec-Université Laval Research Centre, Québec City, QC, Canada.
⁴ Université Laval Cancer Research Centre, Québec City, QC, Canada.
⁵ Department of Cell Biology and Anatomy, Hotchkiss Brain Institute, University of Calgary, Calgary, AB, Canada.
⁶ Department of Immunology, University of Toronto, Toronto, ON, Canada.
⁷ Network Biology Collaborative Centre Flow Cytometry Facility, Sinai Health, Lunenfeld-Tanenbaum Research Institute, Toronto, ON, Canada.
⁸ Alberta Children's Hospital Research Institute, University of Calgary, Calgary, AB, Canada.
⁹ Hotchkiss Brain Institute, University of Calgary, Calgary, AB, Canada.
¹⁰ Department of Laboratory Medicine and Pathobiology, University of Toronto, Toronto, ON, Canada.
¹¹ Division of Anatomy, Department of Surgery, University of Toronto, Toronto, ON, Canada.

Abstract

Introduction: Astrocyte-specific cell surface antigen-2 (ACSA2) has been established as the gold-standard marker for isolating astrocytes via magnetic-activated cell sorting (MACS) or fluorescence-activated cell sorting (FACS) for downstream transcriptomic studies. In a prior study of the astrocyte response to cortical stroke, we used ACSA2-based cell sorting prior to single cell RNA sequencing (scRNAseq). We found a substantial population of ACSA2⁺ cells exhibiting robust microglial gene expression signatures, suggesting contamination of purified astrocyte preparations.

Methods: An intravenous antibody labeling strategy coupled with flow cytometry was used to determine whether contamination originated from circulating immune cells or microglia.

Results: Contaminating cells were identified as CNS-resident microglia that express CD45, CD11b, and ACSA2.

Discussion: These findings caution against the usage of ACSA2 for astrocyte purification without exclusion markers to achieve high-purity astrocyte populations for downstream multi-omics analyses.

Keywords: ACSA2; FACS; MACS; astrocyte; brain cell isolation; scRNA-seq.