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

## TL;DR

ACSA2 is not specific to astrocytes, as it is also expressed by microglia, leading to contamination in astrocyte isolation methods.

## Contribution

The study reveals that ACSA2 is expressed by microglia, challenging its use as a specific astrocyte marker.

## Key findings

- ACSA2+ cells included microglia with strong gene expression signatures.
- Contamination originated from CNS-resident microglia expressing ACSA2, CD45, and CD11b.
- ACSA2 alone is insufficient for high-purity astrocyte isolation.

## Abstract

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.

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

Contaminating cells were identified 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.