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An overview of isolation methods and characterization for TEV and AEEV. Plasma or serum with and without PK was used to test five TEV isolation methods: precipitation (SBI ExoQuick and LabSpinner ExoPRISM); SEC (SBI SmartSEC and Izon qEV); precipitation + purification (SBI ExoQuick Ultra); Silicon Carbide (Norgen) and dUC. AEEV were immunocaptured with a biotinylated antibody against the astrocyte‐specific GLAST antibody. Characterization steps for TEV and AEEV included detection of tetraspanins (CD63, CD81, CD9), syntenin‐1, Alix and calnexin (western blot analysis); particle concentration and quantification (MRPS); tetraspanin bioassays (MSD) and protein concentrations (BCA). Functional assays include cytokine, astrocyte and immune markers. TEV were compared for EV yield and purity, whilst AEEV were compared for purity and recovery. AEEV indicates astrocyte‐enriched extracellular vesicles; ApoE, apolipoprotein E; BCA, bicinchoninic assay; CD14, cluster of differentiation 14; dUC, differential ultracentrifugation; GFAP, glial fibrillary acidic protein; GLAST, glutamate aspartate transporter; IL‐6, interleukin‐6; LBP, lipopolysaccharide‐binding protein; MRPS, microfluidic resistive pulse sensing; MSD, Meso Scale Discovery; PK, proteinase K; SBI, systems biosciences; SEC, size exclusion chromatography; TEV, total EV; <t>TNF,</t> tumor necrosis factor.
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An overview of isolation methods and characterization for TEV and AEEV. Plasma or serum with and without PK was used to test five TEV isolation methods: precipitation (SBI ExoQuick and LabSpinner ExoPRISM); SEC (SBI SmartSEC and Izon qEV); precipitation + purification (SBI ExoQuick Ultra); Silicon Carbide (Norgen) and dUC. AEEV were immunocaptured with a biotinylated antibody against the astrocyte‐specific GLAST antibody. Characterization steps for TEV and AEEV included detection of tetraspanins (CD63, CD81, CD9), syntenin‐1, Alix and calnexin (western blot analysis); particle concentration and quantification (MRPS); tetraspanin bioassays (MSD) and protein concentrations (BCA). Functional assays include cytokine, astrocyte and immune markers. TEV were compared for EV yield and purity, whilst AEEV were compared for purity and recovery. AEEV indicates astrocyte‐enriched extracellular vesicles; ApoE, apolipoprotein E; BCA, bicinchoninic assay; CD14, cluster of differentiation 14; dUC, differential ultracentrifugation; GFAP, glial fibrillary acidic protein; GLAST, glutamate aspartate transporter; IL‐6, interleukin‐6; LBP, lipopolysaccharide‐binding protein; MRPS, microfluidic resistive pulse sensing; MSD, Meso Scale Discovery; PK, proteinase K; SBI, systems biosciences; SEC, size exclusion chromatography; TEV, total EV; TNF, tumor necrosis factor.

Journal: Journal of Extracellular Biology

Article Title: Comparison of Methods for Isolation and Characterization of Total and Astrocyte‐Enriched Extracellular Vesicles From Human Serum and Plasma

doi: 10.1002/jex2.70035

Figure Lengend Snippet: An overview of isolation methods and characterization for TEV and AEEV. Plasma or serum with and without PK was used to test five TEV isolation methods: precipitation (SBI ExoQuick and LabSpinner ExoPRISM); SEC (SBI SmartSEC and Izon qEV); precipitation + purification (SBI ExoQuick Ultra); Silicon Carbide (Norgen) and dUC. AEEV were immunocaptured with a biotinylated antibody against the astrocyte‐specific GLAST antibody. Characterization steps for TEV and AEEV included detection of tetraspanins (CD63, CD81, CD9), syntenin‐1, Alix and calnexin (western blot analysis); particle concentration and quantification (MRPS); tetraspanin bioassays (MSD) and protein concentrations (BCA). Functional assays include cytokine, astrocyte and immune markers. TEV were compared for EV yield and purity, whilst AEEV were compared for purity and recovery. AEEV indicates astrocyte‐enriched extracellular vesicles; ApoE, apolipoprotein E; BCA, bicinchoninic assay; CD14, cluster of differentiation 14; dUC, differential ultracentrifugation; GFAP, glial fibrillary acidic protein; GLAST, glutamate aspartate transporter; IL‐6, interleukin‐6; LBP, lipopolysaccharide‐binding protein; MRPS, microfluidic resistive pulse sensing; MSD, Meso Scale Discovery; PK, proteinase K; SBI, systems biosciences; SEC, size exclusion chromatography; TEV, total EV; TNF, tumor necrosis factor.

Article Snippet: V‐PLEX custom human biomarkers (IL‐6, IL‐8, IL‐10, TNF) , Meso Scale Discovery , K151A9H‐2.

Techniques: Isolation, Clinical Proteomics, Purification, Western Blot, Concentration Assay, Functional Assay, Binding Assay, Size-exclusion Chromatography

Comparison of plasma and serum EV for cytokine markers. IL‐6, IL‐8, IL‐10 and TNF from plasma and serum EV concentrations were measured using V‐PLEX human proinflammatory panel 1 high‐sensitivity assays from MSD. CD9 was measured using R‐PLEX human CD9 (EV) assay. Final concentrations were adjusted to the starting material volume. AU: Human EV Calibrator Standard 1 (to standard curve) = 1000 AU/L. Sample used: age – 33, sex – male, BMI – 25.77. AU indicates arbitrary units; IL, Interleukin; MSD, Meso Scale Discovery; TNF, tumor necrosis factor.

Journal: Journal of Extracellular Biology

Article Title: Comparison of Methods for Isolation and Characterization of Total and Astrocyte‐Enriched Extracellular Vesicles From Human Serum and Plasma

doi: 10.1002/jex2.70035

Figure Lengend Snippet: Comparison of plasma and serum EV for cytokine markers. IL‐6, IL‐8, IL‐10 and TNF from plasma and serum EV concentrations were measured using V‐PLEX human proinflammatory panel 1 high‐sensitivity assays from MSD. CD9 was measured using R‐PLEX human CD9 (EV) assay. Final concentrations were adjusted to the starting material volume. AU: Human EV Calibrator Standard 1 (to standard curve) = 1000 AU/L. Sample used: age – 33, sex – male, BMI – 25.77. AU indicates arbitrary units; IL, Interleukin; MSD, Meso Scale Discovery; TNF, tumor necrosis factor.

Article Snippet: V‐PLEX custom human biomarkers (IL‐6, IL‐8, IL‐10, TNF) , Meso Scale Discovery , K151A9H‐2.

Techniques: Comparison, Clinical Proteomics

Comparison of plasma and serum AEEV immune biomarkers. IL‐6, IL‐8, IL‐10 and TNF‐alpha from plasma and serum AEEV concentrations were measured using V‐PLEX human proinflammatory panel 1 high‐sensitivity assays from MSD. CD9 was measured using R‐PLEX human CD9 (EV) assay. Final concentrations were in 250 µL MPER buffer with 1X PIC regardless of starting volume. AU: Human EV Calibrator Standard 1 (to standard curve) = 1000 AU/L. Sample used: age – 47, sex – male, BMI – 30.81. AEEV indicates astrocyte‐enriched extracellular vesicles; AU, arbitrary units; MSD, Meso Scale Discovery; TNF, tumor necrosis factor.

Journal: Journal of Extracellular Biology

Article Title: Comparison of Methods for Isolation and Characterization of Total and Astrocyte‐Enriched Extracellular Vesicles From Human Serum and Plasma

doi: 10.1002/jex2.70035

Figure Lengend Snippet: Comparison of plasma and serum AEEV immune biomarkers. IL‐6, IL‐8, IL‐10 and TNF‐alpha from plasma and serum AEEV concentrations were measured using V‐PLEX human proinflammatory panel 1 high‐sensitivity assays from MSD. CD9 was measured using R‐PLEX human CD9 (EV) assay. Final concentrations were in 250 µL MPER buffer with 1X PIC regardless of starting volume. AU: Human EV Calibrator Standard 1 (to standard curve) = 1000 AU/L. Sample used: age – 47, sex – male, BMI – 30.81. AEEV indicates astrocyte‐enriched extracellular vesicles; AU, arbitrary units; MSD, Meso Scale Discovery; TNF, tumor necrosis factor.

Article Snippet: V‐PLEX custom human biomarkers (IL‐6, IL‐8, IL‐10, TNF) , Meso Scale Discovery , K151A9H‐2.

Techniques: Comparison, Clinical Proteomics

KEY RESOURCES TABLE

Journal: Cell reports

Article Title: SIV-specific antibodies protect against inflammasome-driven encephalitis in untreated macaques

doi: 10.1016/j.celrep.2024.114833

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: MSD U-PLEX Biomarker Group 1 (NHP) Assays, custom 10-cytokine panel , Meso Scale Discovery , Cat#K15068M.

Techniques: Virus, Clinical Proteomics, Biomarker Discovery, Polymer, RNAscope, Enzyme-linked Immunosorbent Assay, Luciferase, Software