Journal: Journal of Extracellular Vesicles

Article Title: In‐Solution Characterization of Extracellular Vesicles: A New Approach to Evaluating Antibody Binding and Surface Interactions

doi: 10.1002/jev2.70269

Figure Lengend Snippet: Characterization of indicator antibodies by FIDA using different concentrations. A) Taylorgram showing relative fluorescence units (RFU) over time for increasing concentration of anti‐CD63 AF647, 0.88 nM (Rh: 5.24 nm); 1.66 nM (Rh: 5.41 nm); 3.33 nM (Rh: 5.37 nm) and 6.67 nM (Rh: 5.38 nm). B) Taylorgram showing relative fluorescence units (RFU) over time for 0.1–10 nM IgG1‐APC. C) Taylorgram showing relative fluorescence units (RFU) over time for 1–40 nM Cetuximab ALC647.

Article Snippet: EV samples (FLuoEVs, ciMSC‐EVs, Fc‐EVs, and Control EVs) were diluted in assay buffer to the respective concentration based on the particle concentration determined by NTA and/or IFCM followed by incubation with the respective antibodies: mouse anti‐human CD63 AF647 (H5C6, BD Biosciences, Cat. No. 561983, Herlev, Denmark), REA human IgG1 isotype control‐APC (REA293, Miltenyi Biotech, Cat. No. 130‐113‐434, Bergisch Gladbach, Germany) and Cetuximab (ERBITUX, 500 mg/100 mL Cetuximab, Cat. # 06185153) manually labeled with ALC647.

Techniques: Fluorescence, Concentration Assay

Journal: Journal of Extracellular Vesicles

Article Title: In‐Solution Characterization of Extracellular Vesicles: A New Approach to Evaluating Antibody Binding and Surface Interactions

doi: 10.1002/jev2.70269

Figure Lengend Snippet: Binding measurements of increasing concentrations of IgG1‐APC and Cetuximab ALC647 to 1.02 × 10 9 p/mL CD63‐mNG‐Fc EVs. A) Changes of the hydrodynamic radius (Rh) of the complex of CD63‐mNG‐Fc EVs with IgG1‐APC in increasing concentrations of antibody, at 50 mbar ( n = 3). B) Percentage of bound IgG1‐APC by increasing concentrations of antibody to CD63‐mNG‐Fc EVs, at 50 mbar ( n = 3), r 2 = 0.9115. C) Area free/bound IgG1 antibody when incubated with CD63‐mNG‐Fc EVs on increasing concentrations, at 50 mbar ( n = 3), r 2 = 0.9997. D) Changes of the hydrodynamic radius (Rh) of the complex of CD63‐mNG‐Fc EVs with IgG1‐APC in increasing concentrations of antibody, at 100 mbar ( n = 3). E) Percentage of bound IgG1‐APC by increasing concentrations of antibody to CD63‐mNG‐Fc EVs, at 100 mbar ( n = 3), r 2 = 0.8818. F) Area free/bound IgG1 antibody when incubated with CD63‐mNG‐Fc EVs on increasing concentrations, at 100 mbar ( n = 3), r 2 = 0.9996. G) Changes of the hydrodynamic radius (Rh) of the complex of CD63‐mNG‐Fc EVs with Cetuximab in increasing antibody concentrations, at 50 mbar ( n = 3). H) Percentage of bound Cetuximab by increasing concentrations of antibody to CD63‐mNG‐Fc EVs, at 50 mbar ( n = 3), r 2 = 0.9851. I) Area free/bound Cetuximab ALC647 antibody when incubated with CD63‐mNG‐Fc EVs on increasing concentrations, at 50 mbar ( n = 3), r 2 = 0.9950 respectively. J) Changes of the hydrodynamic radius (Rh) of the complex of CD63‐mNG‐Fc EVs with Cetuximab in increasing antibody concentrations, at 100 mbar ( n = 3). K) Percentage of bound Cetuximab antibodies by increasing concentrations of antibody to CD63‐mNG‐Fc EVs, at 100 mbar ( n = 3), r 2 = 0.9537. L) Area free/bound Cetuximab ALC647 antibody when incubated with CD63‐mNG‐Fc EVs on increasing concentrations, at 100 mbar ( n = 3), r 2 = 0.9926, respectively.

Article Snippet: EV samples (FLuoEVs, ciMSC‐EVs, Fc‐EVs, and Control EVs) were diluted in assay buffer to the respective concentration based on the particle concentration determined by NTA and/or IFCM followed by incubation with the respective antibodies: mouse anti‐human CD63 AF647 (H5C6, BD Biosciences, Cat. No. 561983, Herlev, Denmark), REA human IgG1 isotype control‐APC (REA293, Miltenyi Biotech, Cat. No. 130‐113‐434, Bergisch Gladbach, Germany) and Cetuximab (ERBITUX, 500 mg/100 mL Cetuximab, Cat. # 06185153) manually labeled with ALC647.

Techniques: Binding Assay, Incubation

Journal: bioRxiv

Article Title: Dysplastic Epithelial Repair Propagates Chronic Pathology Through the Paracrine Transformation of Pulmonary Fibroblasts

doi: 10.64898/2026.04.02.716135

Figure Lengend Snippet: ( A ) Quantified levels of IL-1α from naïve fibroblast (Naïve fibro), inflammatory fibroblast (Inflam fibro), and ectopic basal cell (Basal) conditioned media as determined by ELISA. ( B ) Saa3 and Ccl2 gene expression by RT-qPCR for fibroblasts cultured on 0.5kPa plates and treated with a 5-fold titration of IL-1α added to control media, including the level determined in (A). ( C ) Graphic illustrating the strategy used to deplete conditioned media of endogenous IL-1α with antibodies, magnetic beads, and column-based separation. ( D ) Saa3 and Ccl2 gene expression by RT-qPCR for fibroblasts cultured on 0.5kPa plates in control or conditioned media depleted using IgG (CTRL IgG or CM IgG) or IL-1α nAb (CTRL nAB, CM nAb) and supplemented with additional IL-1α nAb after depletion (+Spike). [(B) and (D)] Data shows mean fold change ± SD and are pooled from 3 independent experiments (n=3 mice). P values were calculated using either (B) a ratio paired t -test or (D) a one-way ANOVA with post-hoc Turkey multiple comparison test: * P < 0.05, ** P < 0.01, **** P < 0.0001, ns: not significant.

Article Snippet: The working concentration for the following reagents can be found in the text and figure legends: Human IL-1RA Recombinant Protein (IL1Ra) (PeproTech®, 200-01RA), Mouse IL-1 alpha Recombinant Protein, (PeproTech®, 211-11A), Mouse IL-1α Neutralizing Antibody (Invivogen, Anti-mIL-1α-mIgG1 clone 6H7, mIL-1α-mab9-02), Recombinant Mouse IgG1 Isotype Control Antibody (Invivogen, Anti-β-Gal-mIgG1 clone T9C6, bgal-mab9-02), Human TGF-β 1 Recombinant Protein (PeproTech®, 100-21), Human GDF15 Recombinant Protein (PeproTech®, 120-28C), Human VEGF Recombinant Protein (PeproTech®, 450-32), Human OPN Recombinant Protein (PeproTech®, 120-35), Murine CXCL1 Recombinant Protein (PeproTech®, 250-11), Human IGFBP3 Recombinant Protein (PeproTech®, 100-08), Human TNFRSF11B Recombinant Protein (PeproTech®, 450-14), Murine CXCL5 Recombinant Protein (PeproTech®, 250-17), and Muring CCL20 Recombinant Protein (PeproTech®, 250-27).

Techniques: Enzyme-linked Immunosorbent Assay, Gene Expression, Quantitative RT-PCR, Cell Culture, Titration, Control, Magnetic Beads, Comparison