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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 <t>transporter</t> <t>(GLAST,</t> 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.
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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.

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

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.

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