glast (acsa-1) antibody, anti-human/mouse/rat  (Miltenyi Biotec)

Journal: Journal of Extracellular Biology

Article Title: Immunoaffinity‐Based Protocol to Enrich Nervous System Cell‐, Lung Alveolar Cell‐, and Hepatocyte‐Derived Extracellular Vesicles From Human Plasma

doi: 10.1002/jex2.70171

Figure Lengend Snippet: Selection of tissue‐specific surface targets and validation markers. Graphic representation of the tissue and cell type explored in this study. Nervous system EVs have been investigated targeting surface markers specific to neurons, astrocytes and microglia cells. The EV subpopulation from neurons was enriched targeting the growth‐associated protein 43 (GAP43) and neuroligin 3 (NLGN3) as surface markers, while the validation was assessed by measuring the amount of synaptophysin protein (SYP). EVs released from astrocytes and microglia cells were immunoprecipitated using antibodies towards the glutamate aspartate transporter (GLAST, also known as Excitatory amino acid transporter 1‐EAAT1) and the transmembrane protein 119 (TMEM119) respectively and measuring glial fibrillary acidic protein (GFAP) protein for astrocyte EVs, human allograft inflammatory factor 1 (AIF1) and purinergic receptor P2Y12 (P2RY12) for microglia EVs. The human type I cell 56‐kDa protein (HT1‐56) and/or the advanced glycosylation end‐product specific receptor (AGER) were targeted to enrich EVs release from the Alveolar Type 1 lung cells (AT1 cells). The validation was performed by measuring the protein level of human aquaporin 5 (AQP5) and human napsin‐A (NAPSA). Liver EVs released from hepatocytes have been immunocaptured targeting glucose transporter 2 (GLUT2) and/or the asialoglycoprotein receptor 1 (ASGR1), validating the protein level of ASGR1 and cytochrome P450 family 2 subfamily E member 1 (CYP2E1). EVs released from the placenta tissue, specifically from trophoblast cells, have been targeted using placental alkaline phosphatase (PLAP) as surface marker, and measuring the level of cytochrome P450 family 19 subfamily A member 1 (CYP19A1) as validation marker.

Article Snippet: GLAST Glutamate Aspartate Transporter , Miltenyi Biotec (Bergisch Gladbach, Germany) , 130‐118‐984 , Monoclonal , 5.0 , Astrocyte , NS.

Techniques: Selection, Biomarker Discovery, Immunoprecipitation, Glycoproteomics, Marker

Journal: Journal of Extracellular Biology

Article Title: Immunoaffinity‐Based Protocol to Enrich Nervous System Cell‐, Lung Alveolar Cell‐, and Hepatocyte‐Derived Extracellular Vesicles From Human Plasma

doi: 10.1002/jex2.70171

Figure Lengend Snippet: TS‐EV validation by immunoassay. Bar plots show EV subpopulations validated for tissue‐specific markers, comparing the fractions obtained by IP to the isotype control and the total EV. Data are reported as relative average concentration (ng/mL) ± SEM. (A) GAP43 + || NLGN3 + EVs from neurons; (B) GLAST + EVs from astrocytes; (C, D) TMEM119 + EVs from microglia cells; (E–H) HT1‐56 + EVs, (F–I) AGER + EVs and (G–L) HT1‐56 + || AGER + EVs from alveolar type I lung cells; (M–Q) GLUT2 + EVs, (N–R) ASGR1 + EVs, (O–S) GLUT2 + || ASGR1 + EVs (positive for at least one marker) and (P–T) GLUT2 + /ASGR1 + (‘double‐positive’) EVs from hepatocytes.

Article Snippet: GLAST Glutamate Aspartate Transporter , Miltenyi Biotec (Bergisch Gladbach, Germany) , 130‐118‐984 , Monoclonal , 5.0 , Astrocyte , NS.

Techniques: Biomarker Discovery, Control, Concentration Assay, Marker

Journal: Alzheimer's & Dementia

Article Title: Glutamate carboxypeptidase II activation in astrocytes mediates glymphatic impairment and cognitive vulnerability in the aging brain following surgery

doi: 10.1002/alz.71666

Figure Lengend Snippet: Abdominal surgery induces altered perivascular astrocyte reactivity, loss of aquaporin‐4 (AQP4) polarization, and male‐specific alteration of glutamate signaling pathways in the hippocampal astrocytes of aged mice. (A) Immunohistochemistry for glial fibrillary acidic protein (GFAP; green) and Laminin (blue) in hippocampal CA1 region 3 days after surgery; scale bars, 20 µm. Right, quantification of relative GFAP fluorescence intensity in perivascular regions surrounding Laminin + vessels ( n = 6 slices from three mice per group). (B) Left, representative immunohistochemical images for GFAP (green), AQP4 (red), and Laminin (blue) in hippocampal CA1 region; scale bar, 20 µm. Middle, 3D‐rendered reconstructions and high‐magnification images showing AQP4 localization relative to Laminin + vessels and perivascular (PV) astrocytic structures; scale bar, 10 and 4 µm. Right, quantification of AQP4 signal intensity in GFAP + PV regions and GFAP + non‐PV regions ( n = 6 slices from three mice per group). (A, B) ** p < 0.01, *** p < 0.001, determined by unpaired two‐tailed Student's t ‐test. Data represent mean ± SEM. (C) Experimental flow of fluorescence‐activated cell sorting (FACS)‐based hippocampal astrocyte isolation by using a marker (ACSA‐2) 3 days after surgery, followed by single cell RNA sequencing (scRNA‐seq). (D) Uniform manifold approximation and projection (UMAP) clustering of scRNA‐seq data from hippocampal astrocytes (total 9,574 astrocytes) across female‐sham, female‐surgery, male‐sham, and male‐surgery conditions ( n = 3 mice per group). (E) Dot plot showing the expression of homeostatic and reactive astrocyte state marker genes across identified clusters. (F) Proportional distribution of astrocyte clusters across the four conditions. (G) Left, UMAP plot highlighting astrocyte cluster 4 (total 907 astrocytes). Right, gene set enrichment analysis (GSEA) of cluster 4 showing enriched gene ontology (GO) biological process pathways across the three comparisons (female‐sham vs. male‐sham; S, female‐sham vs. female‐surgery, F; and male‐sham vs. male‐surgery, M), showing normalized enrichment score (NES) values. (H) GSEA heatmap of amino acid–related GO biological process pathways in astrocyte cluster 4 across the three group comparisons, showing NES values. (G, H) * p < 0.1, ** p < 0.05, false discovery rate (FDR)‐adjusted p ‐values.

Article Snippet: Cells were subsequently stained for 30 minutes at 4 °C with PE conjugated anti‐mouse ACSA‐2 (cat # 130‐123‐284, Miltenyi Biotec, clone IH3‐18A3, 1:25 dilution), LIVE/DEAD Fixable green dead cell Staining Solution (cat # L34970 , Invitrogen), and Hoechst 33342 (cat # H3570, Invitrogen).

Techniques: Protein-Protein interactions, Immunohistochemistry, Fluorescence, Immunohistochemical staining, Two Tailed Test, FACS, Isolation, Marker, Single Cell, RNA Sequencing, Expressing

Journal: Alzheimer's & Dementia

Article Title: Glutamate carboxypeptidase II activation in astrocytes mediates glymphatic impairment and cognitive vulnerability in the aging brain following surgery

doi: 10.1002/alz.71666

Figure Lengend Snippet: Elevation of hippocampal glutamate and increased astrocyte glutamate carboxypeptidase II (GCPII) activity in aged male mice following abdominal surgery. (A) Glutamate concentration in the hippocampus of male and female mice measured 3 days after surgery ( n = 6 per group). *** p < 0.001, determined by unpaired two‐tailed Student's t ‐test. (B) Demographics of publicly available bulk RNA‐seq datasets of human post mortem brains. Characteristics of the neurotypical controls samples used for the analyses of the relationship between age and FOLH1 , GLS , SLC1A2 , SLC1A3 , and SLC7A11 expression in the hippocampus (top) and the dorsolateral prefrontal cortex (bottom) are shown. (C–G) Scatterplots of adjusted gene expression level (RPKM, reads per kilobase per million mapped reads, adjusted by race and RNA Integrity Number) for FOLH1 (GCPII), GLS , SLC1A2 , SLC1A3 , and SLC7A11 in human post mortem hippocampus (HPC; left panels, N = 194) and dorsolateral prefrontal cortex (DLPFC; right panels, N = 171), stratified by sex (male, green; female, red). Lines represent linear regression fits and Spearman's correlation coefficients ρ and p values are shown above each plot for males and females. (H) Relative Folh1 (GCPII) mRNA expression in hippocampus of male and female mice across young, aged, and aged surgery groups ( n = 12 per group). (I) GCPII activity in hippocampal ACSA‐2 + astrocytes and microglia‐enriched CD11b + cells ( n = 3 per group, pooled hippocampus from two mice per sample). (H, I) * p < 0.05, ** p < 0.01, *** p < 0.001, determined by two‐way analysis of variance (ANOVA) with Tukey's post hoc test. Data represent mean ± SEM.

Article Snippet: Cells were subsequently stained for 30 minutes at 4 °C with PE conjugated anti‐mouse ACSA‐2 (cat # 130‐123‐284, Miltenyi Biotec, clone IH3‐18A3, 1:25 dilution), LIVE/DEAD Fixable green dead cell Staining Solution (cat # L34970 , Invitrogen), and Hoechst 33342 (cat # H3570, Invitrogen).

Techniques: Activity Assay, Concentration Assay, Two Tailed Test, RNA Sequencing, Expressing, Gene Expression