Journal: Cell communication and signaling : CCS

Article Title: Assessment of technical and clinical utility of a bead-based flow cytometry platform for multiparametric phenotyping of CNS-derived extracellular vesicles.

doi: 10.1186/s12964-023-01308-9

Figure Lengend Snippet: Fig. 1 Graphic illustration of the different combinations of sample material and EV isolation protocols applied to the EV Neuro assay. For further details, see text. Abbreviations: HA: primary human astrocytes; GB: glioblastoma; HC: healthy control; MS: multiple sclerosis; MAD: mild cognitive impairment/ Alzheimer’s disease/depression; SEC: size-exclusion chromatography; UC: ultracentrifugation

Article Snippet: MACSPlex EV Kit Neuro, human, reagents were kindly provided by Miltenyi Biotec (Bergisch Gladbach).

Techniques: Isolation, Control, Size-exclusion Chromatography

Journal: Cell communication and signaling : CCS

Article Title: Assessment of technical and clinical utility of a bead-based flow cytometry platform for multiparametric phenotyping of CNS-derived extracellular vesicles.

doi: 10.1186/s12964-023-01308-9

Figure Lengend Snippet: Fig. 2 Characterization of EVs derived from glioblastoma cells and astrocytes. a Size distribution of EVs separated from cell culture supernatant of LN18, LN229, and NCH82 glioblastoma cell lines as well as from primary human astrocytes (HA). b Western blot analysis of CD9, CD63, CD81, Syntenin (EV markers) and Calnexin (non-EV marker) in EVs separated from cell culture supernatant of glioblastoma cells and primary astrocytes. c EV Neuro median signal intensities for LN18, LN229, NCH82, and HA EVs. d Relative signal intensities of values for cell derived EVs shown in (c). Relative signals are calculated by dividing the signal intensity of a target by the sum of intensities revealed from all markers (total intensity) and presented in percentages. Bars reflect representative marker profiles (n = 1–3 biological replicates). Arrow heads indicate markers that appear elevated in cell lines compared to primary astrocytes

Article Snippet: MACSPlex EV Kit Neuro, human, reagents were kindly provided by Miltenyi Biotec (Bergisch Gladbach).

Techniques: Derivative Assay, Cell Culture, Western Blot, Marker

Journal: Cell communication and signaling : CCS

Article Title: Assessment of technical and clinical utility of a bead-based flow cytometry platform for multiparametric phenotyping of CNS-derived extracellular vesicles.

doi: 10.1186/s12964-023-01308-9

Figure Lengend Snippet: Fig. 3 Titration of EVs derived from glioblastoma cells. EV Neuro median signal intensities for increasing total numbers of NCH82 EVs (a) and LN18 EVs (b) as determined by NTA particle count. The number of 2.8 × 107 particles (turquoise) represents the input of 120 µl of pre-cleared cell culture supernatant (10,000 × g) without prior EV enrichment. Other particle inputs (blue and red) were achieved by EV enrichment via dUC. Bars reflect marker profiles of one experiment each

Article Snippet: MACSPlex EV Kit Neuro, human, reagents were kindly provided by Miltenyi Biotec (Bergisch Gladbach).

Techniques: Titration, Derivative Assay, Cell Culture, Marker

Journal: Cell communication and signaling : CCS

Article Title: Assessment of technical and clinical utility of a bead-based flow cytometry platform for multiparametric phenotyping of CNS-derived extracellular vesicles.

doi: 10.1186/s12964-023-01308-9

Figure Lengend Snippet: Fig. 4 Profiling of serum EVs from GB patients and healthy controls. a Relative EV Neuro signal intensities for EVs separated from the serum of GB patients or HC by SEC followed by UC calculated as signal of target divided by the total signal of all markers (in %). Bars represent mean values and error bars indicate the 95% confidence interval. Asterisks mark statistically significant differences between GB and HC. Only significant alterations of targets that were consistently detected above background in all individuals are marked. *=p < .05, **=p < .01, ***=p < .001. (b) tSNE on data from (a) stratified by condition (color). c Heatmap visualization of selected targets from (a) including hierarchical clustering for targets as well as subjects

Article Snippet: MACSPlex EV Kit Neuro, human, reagents were kindly provided by Miltenyi Biotec (Bergisch Gladbach).

Techniques:

Journal: Cell communication and signaling : CCS

Article Title: Assessment of technical and clinical utility of a bead-based flow cytometry platform for multiparametric phenotyping of CNS-derived extracellular vesicles.

doi: 10.1186/s12964-023-01308-9

Figure Lengend Snippet: Fig. 6 Comparison of plasma EVs from MS patients in a relapse versus in a stable disease phase. Relative EV Neuro signal intensities as signal of target divided by the total signal of all markers (in %) for immuno-affinity captured CD63+EVs (a) and CD81+EVs (b). Bars represent mean values and error bars indicate the 95% confidence interval. Asterisks mark statistically significant differences between stable phase and disease relapse. Only significant alterations of targets that were consistently detected above background in all individuals are marked. *=p < .05, **=p < .01

Article Snippet: MACSPlex EV Kit Neuro, human, reagents were kindly provided by Miltenyi Biotec (Bergisch Gladbach).

Techniques: Comparison, Clinical Proteomics

Journal: Cell communication and signaling : CCS

Article Title: Assessment of technical and clinical utility of a bead-based flow cytometry platform for multiparametric phenotyping of CNS-derived extracellular vesicles.

doi: 10.1186/s12964-023-01308-9

Figure Lengend Snippet: Fig. 7 Phenotyping of plasma EVs from MAD patients and comparison of all plasma samples. a Relative EV Neuro signal intensities for EVs separated from the plasma of MAD patients by immuno-affinity capture (anti-CD81) and magnetic separation. Bars represent mean values and error bars indicate the 95% confidence intervals (n = 8 patients). b tSNE of relative signal intensities from all plasma-derived EV samples analyzed (MS, MAD, HC) stratified by condition (color) and isolation (shape). c Heatmap visualization of relative signals for selected targets from all plasma samples analyzed including hierarchical clustering for targets as well as subjects (BCL = lab of blood collection)

Article Snippet: MACSPlex EV Kit Neuro, human, reagents were kindly provided by Miltenyi Biotec (Bergisch Gladbach).

Techniques: Clinical Proteomics, Comparison, Derivative Assay, Isolation