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

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.