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.