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Multiplex bead-based EV flow cytometry assay for surface markers. EVs enriched from UHPi and IVIg by SEC and dUC. Surface marker profiles were measured using Miltenyi MACSPlex EV kit IO with MESF-calibrated flow cytometry. (A) Heatmap and hierarchical clustering of MACSPlex markers in UHPi EVs, IVIg EVs, and bead/antibody controls (left). Median APC intensity (MESF, background-subtracted) of mixed tetraspanin antibodies (CD9/CD81/CD63) on EVs captured by 39 marker beads (middle/right). (B) Enlarged values for the tetraspanins (CD9, CD63, and CD81), HLA markers (HLA-ABC and HLA-DR, DP,DQ), platelet markers (CD42a, CD41b, and CD62p), and stemness markers (CD29, ROR1, CD24, CD326, CD133/1). dUC, differential ultracentrifugation; EV, extracellular vesicles; Human Leukocyte Antigens; HLA; IVIg, intravenous immunoglobulin; MESF, Molecular Equivalents of Soluble Fluorophore; SEC, size-exclusion chromatography; UHPi, individual unprocessed human plasma.

Journal: bioRxiv

Article Title: Translational Opportunity of Engineered IFNγ-eEVs Through Targeted Inhibition of JAK/STAT1 Signaling, Mimicking IVIg Therapy

doi: 10.64898/2026.04.29.721601

Figure Lengend Snippet: Multiplex bead-based EV flow cytometry assay for surface markers. EVs enriched from UHPi and IVIg by SEC and dUC. Surface marker profiles were measured using Miltenyi MACSPlex EV kit IO with MESF-calibrated flow cytometry. (A) Heatmap and hierarchical clustering of MACSPlex markers in UHPi EVs, IVIg EVs, and bead/antibody controls (left). Median APC intensity (MESF, background-subtracted) of mixed tetraspanin antibodies (CD9/CD81/CD63) on EVs captured by 39 marker beads (middle/right). (B) Enlarged values for the tetraspanins (CD9, CD63, and CD81), HLA markers (HLA-ABC and HLA-DR, DP,DQ), platelet markers (CD42a, CD41b, and CD62p), and stemness markers (CD29, ROR1, CD24, CD326, CD133/1). dUC, differential ultracentrifugation; EV, extracellular vesicles; Human Leukocyte Antigens; HLA; IVIg, intravenous immunoglobulin; MESF, Molecular Equivalents of Soluble Fluorophore; SEC, size-exclusion chromatography; UHPi, individual unprocessed human plasma.

Article Snippet: EVs isolated from UHPi, UHPp, and IVIg were subjected to immune profiling for 39 known EV surface proteins by flow cytometry using the human MACSPlex EV kit IO (Miltenyi Biotec, Auburn, CA) and detected by flow cytometry (Cytek Aurora, Cytek Biosciences) with fluorescence calibration using Molecular Equivalents of Soluble Fluorophore (MESF) standards (Quantum APC MESF Beads, Bangs Laboratories) as previously described ( ; ).

Techniques: Multiplex Assay, Flow Cytometry, Marker, Size-exclusion Chromatography, Clinical Proteomics