Anti-ACSA-2 defines a novel monoclonal antibody for prospective isolation of living neonatal and adult astrocytes

Abstract

Astrocytes are the most abundant cell type of the central nervous system and cover a broad range of functionalities. We report here the generation of a novel monoclonal antibody, anti-astrocyte cell surface antigen-2 (Anti-ACSA-2). Flow cytometry, immunohistochemistry and immunocytochemistry revealed that Anti-ACSA-2 reacted specifically with a not yet identified glycosylated surface molecule of murine astrocytes at all developmental stages. It did not show any labeling of non-astroglial cells such as neurons, oligodendrocytes, NG2 cells, microglia, endothelial cells, leukocytes, or erythrocytes. Co-labeling studies of GLAST and ACSA-2 showed largely overlapping expression. However, there were also notable differences in protein expression levels and frequencies of single-positive subpopulations of cells in some regions of the CNS such as cerebellum, most prominently at early postnatal stages. In the neurogenic niches, the dentate gyrus of the hippocampus and the subventricular zone (SVZ), again a general overlap with slight differences in expression levels were observed. ACSA-2 was unlike GLAST not sensitive to papain-based tissue dissociation and allowed for a highly effective, acute, specific, and prospective purification of viable astrocytes based on a new rapid sorting procedure using Anti-ACSA-2 directly coupled to superparamagnetic MicroBeads. In conclusion, ACSA-2 appears to be a new surface marker for astrocytes, radial glia, neural stem cells and bipotent glial progenitor cells which opens up the possibility of further dissecting the characteristics of astroglial subpopulations and lineages.

Inducible cell-specific mouse models for paired epigenetic and transcriptomic studies of microglia and astroglia

“…As an initial validation of the model, Aldh1l1-NuTRAP mice were systemically delivered Tam for 5 consecutive days and a week after induction, brains were dissected for flow cytometry (FC) and immunohistochemistry (IHC) analyses. Single-cell suspensions of brains immunostained with ACSA-2 antibody, a pan-astrocytic marker 15 , revealed a distinct EGFP + cell population present in the Aldh1l1-NuTRAP brains but not in the cre negative counterparts, consistent with the reported 10-20% astroglial cellularity in the rodent brain 16, 17 , Almost the entirety of the EGFP + cell population co-expressed ACSA-2, supporting that cre-mediated recombination upon Tam induction specifically targeted astrocytes (Figure 1A-B) .…”

“…Subsequent gating based on CD11b or ACSA-2 expression was done with 640 nm laser and 676/629 filter, or with 488 nm laser and 740 LP filter combinations, respectively. The antibodies used were anti-mouse CD11b: APC (#17-0112, clone M1/70) (eBioscience, San Diego, CA), and ACSA-2: PE-Vio770 (#130-116-246, Milteny Biotec) 15 . Isotype controls for each antibody and unstained cells were used for proper post-color compensation (Supplemental Figures 14-15) .…”

Concurrent cell type–specific isolation and profiling of mouse brains in inflammation and Alzheimer’s disease

“…These include the well-established antibodies against CD45 (36) and O4 (37,38), the astrocyte-specific anti-ACSA-2 antibody that has been recently validated by ourselves and independent groups (27,39,40), (Supplemental Figure 2), and the anti-CD49a antibody ( Figure 1B, Figure 2, and Supplemental Figure 2). Employing the commonly used ALDH1l1-EGFP reporter (41) believed to mark the majority of astrocytes, we found that the anti-ACSA-2 antibody labeled all GFP + astrocytes in addition to a sizable GFP -astrocyte population (Supplemental Figure 3A), suggesting that this reporter line does not mark all astrocytes in adult brains.…”

GLP-1R agonist NLY01 reduces retinal inflammation, astrocyte reactivity, and retinal ganglion cell death secondary to ocular hypertension

“…S1B-C). Astrocytes and Müller cells were isolated using ASCA2+ selection, as reported previously (Kantzer et al, 2017). By 14 days post-injection, ACSA2+ cells exhibited increased levels of pan reactive and A1 astrocyte specific markers ( Fig.…”