ORIGINAL RESEARCH article
Front. Cell. Neurosci.
Sec. Cellular Neuropathology
This article is part of the Research TopicSingle-Cell Approaches in Unraveling Central Nervous System Pathologies: Insights into Disease Mechanisms and Therapeutic TargetsView all articles
ACSA-2 is not astrocyte-specific: implications for cell sorting strategies in the rodent brain
Provisionally accepted
Emerson Daniele1
Gabriel Khelifi2
Daniel Beretta3Boyan K Tsankov4KW Annie Bang5
Chiara Beretta3
Dana Philpott4
Samer M. Hussein2
Maryam Faiz3,6*- 1University of Toronto Institute of Medical Science, Toronto, Canada
- 2Universite Laval, Québec City, Canada
- 3University of Calgary Cumming School of Medicine, Calgary, Canada
- 4University of Toronto, Toronto, Canada
- 5Lunenfeld-Tanenbaum Research Institute, Toronto, Canada
- 6Department of Surgery, University of Toronto, Toronto, Canada
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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, in which we used ACSA2-based cell sorting prior to single cell RNA sequencing (scRNAseq), we found a substantial population of ACSA2+ cells that exhibited robust microglial gene expression signatures suggesting contamination of purified astrocyte preparations. To determine whether this contamination originated from circulating immune cells or microglia, we leveraged an intravenous antibody labeling strategy coupled with flow cytometry. This approach identified the contaminating cells as CNS-resident microglia that express CD45, CD11b, and ACSA2. 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: ACSA-2, astrocyte, brain cell isolation, FACS, MACs, ScRNA-seq
Received: 29 Nov 2025; Accepted: 23 Jan 2026.
Copyright: © 2026 Daniele, Khelifi, Beretta, Tsankov, Bang, Beretta, Philpott, Hussein and Faiz. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) or licensor are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.
* Correspondence: Maryam Faiz
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