Journal: Journal of Extracellular Vesicles

Article Title: Unravelling Ligand Conjugation Performance in Extracellular Vesicles: A Quantitative Assessment of Lipid, Protein, and Membrane Modifications

doi: 10.1002/jev2.70174

Figure Lengend Snippet: The impact of ligand conjugation on cell uptake of mEVs‐Tf. (a) Schematic illustration of the principle for evaluating the correlation between ligand conjugation and cell uptake. (b) Confocal fluorescence microscopy analysis of the cell uptake of DiD‐labelled blank mEVs and the transferrin conjugated counterparts in the presence or absence of 50 mM free transferrin, respectively. HepG2 cells were incubated with DiD‐labelled (red) mEV‐Tf for 12 h, and stained with Hoechst 33342 (blue) for cell nuclei imaging. (c) Lysosome colocalization analysis of DiD‐labelled blank mEVs and mEVs‐Tf engineered by different methods. HepG2 cells were incubated with DiD‐labelled (red) mEV samples for 24 h, and stained with Hoechst 33342 (blue) and LysoTracker (green) for cell nuclei and lysosome imaging, respectively. (d) Semi‐quantitative analysis of the colocalization degree between mEVs and lysosomes using the Manders' colocalization coefficients M1 and M2. (e) Representative dot‐plots between particle size and ligand number of the mEVs‐Tf‐CF488 engineered by lipid modification (panel‐i), protein modification (panel‐ii) and membrane insertion (panel‐iii), respectively. The transferrin concentration was 50 µM for all three samples. (f), (g) Percentage of Tf‐CF488 positive particles (f) and ligand density (g) of the mEVs‐Tf‐CF488 engineered by different methods with increasing input concentrations of Tf‐CF488. (h) FCM analysis of the impact of ligand density on the cell uptake degree of the DiD‐labelled mEVs‐Tf‐CF488 engineered by lipid modification (panel‐i), protein modification (panel‐ii) and membrane insertion (panel‐iii), respectively. (i,j) The relationship between cell fluorescence intensity (FL‐DiD) and input concentration of Tf‐CF488 (i), and ligand density (j) for DiD‐labelled mEVs‐Tf‐CF488 engineered by different methods.

Article Snippet: The concentration of accessible transferrin conjugated on mEVs was measured by a human transferrin ELISA kit (MultiSciences).

Techniques: Conjugation Assay, Fluorescence, Microscopy, Incubation, Staining, Imaging, Modification, Membrane, Concentration Assay

Journal: Journal of Extracellular Vesicles

Article Title: Unravelling Ligand Conjugation Performance in Extracellular Vesicles: A Quantitative Assessment of Lipid, Protein, and Membrane Modifications

doi: 10.1002/jev2.70174

Figure Lengend Snippet: Impact of ligand conjugation on MFGE8 accessibility and surface‐ligand distribution homogeneity of transferrin. (a) Schematic illustration of ligand conjugation effects on the cell binding behaviour of mEVs‐Tf. (b) FCM analysis of the MFGE8‐mediated cell uptake of mEVs. HepG2 cells were treated with DiD‐labelled blank mEVs with (blue) or without (red) labelling against MFGE8 antibodies. (c) Relationships between particle size and the immunofluorescence intensity of MFGE8 for blank mEVs (panel‐i) and the transferrin‐conjugated mEVs engineered by lipid modification (panel‐ii), protein modification (panel‐iii) and membrane insertion (panel‐iv), respectively. (d) The percentage of MFGE8‐positive particles (left y‐axis) and the immunofluorescence intensity (right y‐axis) of the mEV samples. (e) Correlation between the fluorescence intensity of CF488 versus CF594 for individual mEVs‐Tf‐CF488 (panel‐i) and mEVs‐Tf‐CF488&Tf‐CF594 engineered by lipid modification (panel‐ii), protein modification (panel‐iii) and membrane insertion (panel‐iv), respectively. (f) The percentage of CF594‐positive particles (left y‐axis) and the relative fluorescence intensity of CF594 to CF488 (right y‐axis) of the mEV samples. Note: In these experiments, the concentrations of sPLD, TCEP and DSPE‐PEG‐MAL for lipid‐, protein‐ and membrane‐based ligand conjugation were 10 U/mL, 2 and 1.25 mM, respectively.

Article Snippet: The concentration of accessible transferrin conjugated on mEVs was measured by a human transferrin ELISA kit (MultiSciences).

Techniques: Conjugation Assay, Binding Assay, Immunofluorescence, Modification, Membrane, Fluorescence

Journal: Journal of Extracellular Vesicles

Article Title: Unravelling Ligand Conjugation Performance in Extracellular Vesicles: A Quantitative Assessment of Lipid, Protein, and Membrane Modifications

doi: 10.1002/jev2.70174

Figure Lengend Snippet: In vivo cancer targeting performance of ligand‐conjugated mEVs. (a) In vivo fluorescence images of the cancer‐bearing mice injected with DiD‐labelled blank mEVs, and mEVs‐Tf engineered by distinct methods at different time points. (b) Time‐dependent variation in radiant efficiency of the tumour site of the mice treated with various mEV formulations. (c) Ex vivo fluorescence images of tumours and other organs excised from the treated mice at 24 h post‐injection. (d) Radiant efficiency of the tumours and other organs based on the ex vivo fluorescence images. (e) Schematic illustration of the principle for characterizing the IgG and IgM adsorption on mEVs after serum incubation. (f), (g) Representative distributions and the bivariate dot‐plots of the particle size and the immunofluorescence intensity of IgG (f) and IgM (g) of the mouse serum‐incubated blank mEVs (panel‐i), and the transferrin‐conjugated counterparts engineered by lipid modification (panel‐ii), protein modification (panel‐iii) and membrane insertion (panel‐iv), respectively. (h)–(j) Immunolabelling ratio (h), immunofluorescence intensity (i), and particle size (j) of the mouse serum‐incubated mEV samples. (k) The ELISA measurement of the accessible transferrin concentration of the mEV samples before and after serum incubation.

Article Snippet: The concentration of accessible transferrin conjugated on mEVs was measured by a human transferrin ELISA kit (MultiSciences).

Techniques: In Vivo, Fluorescence, Injection, Ex Vivo, Adsorption, Incubation, Immunofluorescence, Modification, Membrane, Enzyme-linked Immunosorbent Assay, Concentration Assay

Journal: PLoS ONE

Article Title: The Influence of Tobacco Smoke on Protein and Metal Levels in the Serum of Women during Pregnancy

doi: 10.1371/journal.pone.0161342

Figure Lengend Snippet: Concentration of α1-antitrypsin, α2-macroglobulin, α1-acid glycoprotein and total protein concentration in the serum of women during each trimester of pregnancy.

Article Snippet: ELISA tests were used to determine α1-antitrypsin (α1-Antitrypsin Clearance ELISA, Immundiagnostik, No: K6752), α1-acid glycoprotein (Human alpha-1-Acid Glycoprotein ELISA kit, AssayPro No: EG5001-1), α2-macroglobulin (α2-macroglobulin ELISA Kit, Immundiagnostik, No: K6610A) and transferrin (AssayMax Human Transferrin ELISA Kit, AssayPro, No: ET2105-1) levels.

Techniques: Concentration Assay, Protein Concentration

Journal: PLoS ONE

Article Title: The Influence of Tobacco Smoke on Protein and Metal Levels in the Serum of Women during Pregnancy

doi: 10.1371/journal.pone.0161342

Figure Lengend Snippet: Correlations between the concentrations of total protein, serum proteins, α1-antitrypsin, α1-acid glycoprotein, α2-macroglobulin, transferrin, with the Zn/Cd ratio and cotinine, Cd, and Zn concentrations in pregnant smoking women [n = 10] in the 3rd trimester of pregnancy.

Article Snippet: ELISA tests were used to determine α1-antitrypsin (α1-Antitrypsin Clearance ELISA, Immundiagnostik, No: K6752), α1-acid glycoprotein (Human alpha-1-Acid Glycoprotein ELISA kit, AssayPro No: EG5001-1), α2-macroglobulin (α2-macroglobulin ELISA Kit, Immundiagnostik, No: K6610A) and transferrin (AssayMax Human Transferrin ELISA Kit, AssayPro, No: ET2105-1) levels.

Techniques:

Journal: Communications Biology

Article Title: HSPA4 restrains transferrin in dopaminergic neurons to attenuate ferroptosis in a Parkinson’s disease model

doi: 10.1038/s42003-025-08854-7

Figure Lengend Snippet: A HSPA4 colocalizes with transferrin in cells. The expression of HSPA4 (green) and transferrin (TF; red) in SH-SY5Y cells was analyzed by immunofluorescence analysis, and DAPI (blue) was used to stain the nucleus. Scale bar = 5 μm. B Intracellular transferrin was increased by HSPA4. SH-SY5Y cells were transfected with the plasmid expressing Hspa4 . Twenty-four hours post-transfection, the cells were subjected to immunofluorescence analysis using an anti-transferrin antibody. DAPI was used to stain the nucleus. Scale bar = 50 μm. C Quantification of the relative fluorescence intensity of transferrin, as shown in ( B ). n = 5 biologically independent samples. D HSPA4 sequesters transferrin in SH-SY5Y cells. SH-SY5Y cells were transfected with a plasmid expressing Hspa4 . Twenty-four hours post-transfection, the cells and cell culture media were collected, and HSPA4 expression was analyzed by western blot. Actin (left panel) and Coomassie blue staining (right panel) were used as loading controls. E HSPA4 reduces extracellular transferrin. SH-SY5Y cells were transfected with a plasmid expressing Hspa4 . Twenty-four hours post-transfection, the cell culture media were collected, and transferrin was measured with an ELISA kit. n = 4 biologically independent samples. F HSPA4 decreases the level of intracellular Fe 2+ . SH-SY5Y cells were transfected with a plasmid expressing Hspa4 . Twenty-four hours post-transfection, the cells were harvested for analysis of Fe 2+ . n = 3 biologically independent samples. EV: empty vector; TF: transferrin. All data are presented as means ± SEM. Statistical significance was determined by unpaired Student’s t -test.

Article Snippet: Transferrin in the cell culture medium was measured with a commercial ELISA kit (EK1201; MultiScience).

Techniques: Expressing, Immunofluorescence, Staining, Transfection, Plasmid Preparation, Fluorescence, Cell Culture, Western Blot, Enzyme-linked Immunosorbent Assay