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a , Nine EV-sorting domains and nine Fc-binding domains used in different combinations, as illustrated by the examples in the centre, for the screen to optimize Fc-EVs. b , IFC of transient transfection of HEK293T cells with nine different EVs sorting domains, intraluminally fused via the N- or C-terminal to the fluorescent protein mNG (TNFR only have one intraluminal terminus, C-terminus). Wt, wild type. c , IFC comparison of engineered mNG EVs from cells in both configurations based on events in conditioned media. d , IFC analysis of the expression of the Fc binder based on the mean fluorescence intensity (MFI) of mNG for the top candidate combinations of different Fc binders (z, 4z, protein A) with different EVs sorting domains (CD63, CD9, <t>CD81,</t> TNFR). e , IFC analysis of the expression of the Fc binder based on the percentage of double positive (APC Abs binding mNG-Fc-EVs) for the top candidate combinations of different Fc binders (z, 4z, protein A) with different EV-sorting domains (CD63, CD9, CD81, TNFR). Other combinations can be found in Supplementary Fig. . Illustrations created with BioRender.com . GAPDH, first 148 amino acids (aa) of glyceraldehyde-3-phosphate dehydrogenase; ARRDC1, arrestin domain-containing protein; ApoE lipid binding sites (v1 corresponds to 355 aa, v2 corresponds to 1,009 aa); z domain, synthetic Fc region-binding domain originated from the B domain of protein A ; 4z, four repetitive z domains; MRP4, multidrug resistance protein 4; PIP, prolactin-induced protein; hFcγr, human Fc gamma receptor. Panels d and e are shown as mean ± s.d. n = 3 biological replicates. Statistical significance was calculated using two-way ANOVA with Dunnett’s post-test compared with each value; P values are indicated above the plots of d and e .
Cd81, supplied by Miltenyi Biotec, used in various techniques. Bioz Stars score: 95/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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a , Nine EV-sorting domains and nine Fc-binding domains used in different combinations, as illustrated by the examples in the centre, for the screen to optimize Fc-EVs. b , IFC of transient transfection of HEK293T cells with nine different EVs sorting domains, intraluminally fused via the N- or C-terminal to the fluorescent protein mNG (TNFR only have one intraluminal terminus, C-terminus). Wt, wild type. c , IFC comparison of engineered mNG EVs from cells in both configurations based on events in conditioned media. d , IFC analysis of the expression of the Fc binder based on the mean fluorescence intensity (MFI) of mNG for the top candidate combinations of different Fc binders (z, 4z, protein A) with different EVs sorting domains (CD63, CD9, <t>CD81,</t> TNFR). e , IFC analysis of the expression of the Fc binder based on the percentage of double positive (APC Abs binding mNG-Fc-EVs) for the top candidate combinations of different Fc binders (z, 4z, protein A) with different EV-sorting domains (CD63, CD9, CD81, TNFR). Other combinations can be found in Supplementary Fig. . Illustrations created with BioRender.com . GAPDH, first 148 amino acids (aa) of glyceraldehyde-3-phosphate dehydrogenase; ARRDC1, arrestin domain-containing protein; ApoE lipid binding sites (v1 corresponds to 355 aa, v2 corresponds to 1,009 aa); z domain, synthetic Fc region-binding domain originated from the B domain of protein A ; 4z, four repetitive z domains; MRP4, multidrug resistance protein 4; PIP, prolactin-induced protein; hFcγr, human Fc gamma receptor. Panels d and e are shown as mean ± s.d. n = 3 biological replicates. Statistical significance was calculated using two-way ANOVA with Dunnett’s post-test compared with each value; P values are indicated above the plots of d and e .
Anti Human Cd81 Apc Conjugated Antibodies, supplied by Miltenyi Biotec, used in various techniques. Bioz Stars score: 95/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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a , Nine EV-sorting domains and nine Fc-binding domains used in different combinations, as illustrated by the examples in the centre, for the screen to optimize Fc-EVs. b , IFC of transient transfection of HEK293T cells with nine different EVs sorting domains, intraluminally fused via the N- or C-terminal to the fluorescent protein mNG (TNFR only have one intraluminal terminus, C-terminus). Wt, wild type. c , IFC comparison of engineered mNG EVs from cells in both configurations based on events in conditioned media. d , IFC analysis of the expression of the Fc binder based on the mean fluorescence intensity (MFI) of mNG for the top candidate combinations of different Fc binders (z, 4z, protein A) with different EVs sorting domains (CD63, CD9, <t>CD81,</t> TNFR). e , IFC analysis of the expression of the Fc binder based on the percentage of double positive (APC Abs binding mNG-Fc-EVs) for the top candidate combinations of different Fc binders (z, 4z, protein A) with different EV-sorting domains (CD63, CD9, CD81, TNFR). Other combinations can be found in Supplementary Fig. . Illustrations created with BioRender.com . GAPDH, first 148 amino acids (aa) of glyceraldehyde-3-phosphate dehydrogenase; ARRDC1, arrestin domain-containing protein; ApoE lipid binding sites (v1 corresponds to 355 aa, v2 corresponds to 1,009 aa); z domain, synthetic Fc region-binding domain originated from the B domain of protein A ; 4z, four repetitive z domains; MRP4, multidrug resistance protein 4; PIP, prolactin-induced protein; hFcγr, human Fc gamma receptor. Panels d and e are shown as mean ± s.d. n = 3 biological replicates. Statistical significance was calculated using two-way ANOVA with Dunnett’s post-test compared with each value; P values are indicated above the plots of d and e .
Anti Human Cd81, supplied by Miltenyi Biotec, used in various techniques. Bioz Stars score: 95/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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a , Nine EV-sorting domains and nine Fc-binding domains used in different combinations, as illustrated by the examples in the centre, for the screen to optimize Fc-EVs. b , IFC of transient transfection of HEK293T cells with nine different EVs sorting domains, intraluminally fused via the N- or C-terminal to the fluorescent protein mNG (TNFR only have one intraluminal terminus, C-terminus). Wt, wild type. c , IFC comparison of engineered mNG EVs from cells in both configurations based on events in conditioned media. d , IFC analysis of the expression of the Fc binder based on the mean fluorescence intensity (MFI) of mNG for the top candidate combinations of different Fc binders (z, 4z, protein A) with different EVs sorting domains (CD63, CD9, <t>CD81,</t> TNFR). e , IFC analysis of the expression of the Fc binder based on the percentage of double positive (APC Abs binding mNG-Fc-EVs) for the top candidate combinations of different Fc binders (z, 4z, protein A) with different EV-sorting domains (CD63, CD9, CD81, TNFR). Other combinations can be found in Supplementary Fig. . Illustrations created with BioRender.com . GAPDH, first 148 amino acids (aa) of glyceraldehyde-3-phosphate dehydrogenase; ARRDC1, arrestin domain-containing protein; ApoE lipid binding sites (v1 corresponds to 355 aa, v2 corresponds to 1,009 aa); z domain, synthetic Fc region-binding domain originated from the B domain of protein A ; 4z, four repetitive z domains; MRP4, multidrug resistance protein 4; PIP, prolactin-induced protein; hFcγr, human Fc gamma receptor. Panels d and e are shown as mean ± s.d. n = 3 biological replicates. Statistical significance was calculated using two-way ANOVA with Dunnett’s post-test compared with each value; P values are indicated above the plots of d and e .
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Image Search Results


a , Nine EV-sorting domains and nine Fc-binding domains used in different combinations, as illustrated by the examples in the centre, for the screen to optimize Fc-EVs. b , IFC of transient transfection of HEK293T cells with nine different EVs sorting domains, intraluminally fused via the N- or C-terminal to the fluorescent protein mNG (TNFR only have one intraluminal terminus, C-terminus). Wt, wild type. c , IFC comparison of engineered mNG EVs from cells in both configurations based on events in conditioned media. d , IFC analysis of the expression of the Fc binder based on the mean fluorescence intensity (MFI) of mNG for the top candidate combinations of different Fc binders (z, 4z, protein A) with different EVs sorting domains (CD63, CD9, CD81, TNFR). e , IFC analysis of the expression of the Fc binder based on the percentage of double positive (APC Abs binding mNG-Fc-EVs) for the top candidate combinations of different Fc binders (z, 4z, protein A) with different EV-sorting domains (CD63, CD9, CD81, TNFR). Other combinations can be found in Supplementary Fig. . Illustrations created with BioRender.com . GAPDH, first 148 amino acids (aa) of glyceraldehyde-3-phosphate dehydrogenase; ARRDC1, arrestin domain-containing protein; ApoE lipid binding sites (v1 corresponds to 355 aa, v2 corresponds to 1,009 aa); z domain, synthetic Fc region-binding domain originated from the B domain of protein A ; 4z, four repetitive z domains; MRP4, multidrug resistance protein 4; PIP, prolactin-induced protein; hFcγr, human Fc gamma receptor. Panels d and e are shown as mean ± s.d. n = 3 biological replicates. Statistical significance was calculated using two-way ANOVA with Dunnett’s post-test compared with each value; P values are indicated above the plots of d and e .

Journal: Nature Biomedical Engineering

Article Title: Antibody-displaying extracellular vesicles for targeted cancer therapy

doi: 10.1038/s41551-024-01214-6

Figure Lengend Snippet: a , Nine EV-sorting domains and nine Fc-binding domains used in different combinations, as illustrated by the examples in the centre, for the screen to optimize Fc-EVs. b , IFC of transient transfection of HEK293T cells with nine different EVs sorting domains, intraluminally fused via the N- or C-terminal to the fluorescent protein mNG (TNFR only have one intraluminal terminus, C-terminus). Wt, wild type. c , IFC comparison of engineered mNG EVs from cells in both configurations based on events in conditioned media. d , IFC analysis of the expression of the Fc binder based on the mean fluorescence intensity (MFI) of mNG for the top candidate combinations of different Fc binders (z, 4z, protein A) with different EVs sorting domains (CD63, CD9, CD81, TNFR). e , IFC analysis of the expression of the Fc binder based on the percentage of double positive (APC Abs binding mNG-Fc-EVs) for the top candidate combinations of different Fc binders (z, 4z, protein A) with different EV-sorting domains (CD63, CD9, CD81, TNFR). Other combinations can be found in Supplementary Fig. . Illustrations created with BioRender.com . GAPDH, first 148 amino acids (aa) of glyceraldehyde-3-phosphate dehydrogenase; ARRDC1, arrestin domain-containing protein; ApoE lipid binding sites (v1 corresponds to 355 aa, v2 corresponds to 1,009 aa); z domain, synthetic Fc region-binding domain originated from the B domain of protein A ; 4z, four repetitive z domains; MRP4, multidrug resistance protein 4; PIP, prolactin-induced protein; hFcγr, human Fc gamma receptor. Panels d and e are shown as mean ± s.d. n = 3 biological replicates. Statistical significance was calculated using two-way ANOVA with Dunnett’s post-test compared with each value; P values are indicated above the plots of d and e .

Article Snippet: In brief, 2.5 × 10 8 EVs (NTA-based particles) were stained (unless indicated otherwise) in a total volume of 25 μl with 8 nM of fluorescent antibodies against the tetraspanins CD9, CD63 and CD81 or recombinant antibodies (isotypes control) (REA(S))-APC human IgG isotype control antibodies (Miltenyi Biotec).

Techniques: Binding Assay, Transfection, Comparison, Expressing, Fluorescence