Journal: Journal of Extracellular Vesicles

Article Title: Snorkel‐tag based affinity chromatography for recombinant extracellular vesicle purification

doi: 10.1002/jev2.12523

Figure Lengend Snippet: Schematic representation of the Snorkel‐tag genetically fused to the C‐terminus of CD81 on cell and EV surface membranes. Created with BioRender.com.

Article Snippet: For quantification of cellular uptake of StEVAC purified EVs, equal number of eluted EVs from CD81‐Snorkel‐tag, WT and CD81‐Snorkel‐tag were labelled with CLIP‐substrate (CLIP‐Surface™ 647; Catalogue #S9234, NEB and/or CLIP‐Surface™ 488; Catalogue #S9232, NEB) with a final dilution of 1:100,000 was added and incubated for 1 h at 95% humidity, 5% CO 2 and 37°C.

Techniques:

Journal: Journal of Extracellular Vesicles

Article Title: Snorkel‐tag based affinity chromatography for recombinant extracellular vesicle purification

doi: 10.1002/jev2.12523

Figure Lengend Snippet: Expression of CD81‐Snorkel‐tag in HeLa cells results in accessibility of all parts of the Snorkel‐tag. (a) Schematic illustration of snorkel‐tag components fused to C‐termini of CD81. (b) Flow cytometric analysis of HeLa‐WT and HeLa‐CD81‐Snorkel‐tag stable cells stained with CLIP membrane impermeable substrate CLIP‐647. (c) Western blot of HA‐tag, FLAG‐tag, CLIP‐tag, CD81 and beta‐actin for WT and CD81‐Snorkel‐tag cell lines. Beta‐actin was used as loading control. (d) Fluorescent images of fixed WT and CD81‐Snorkel‐tag cells. Right two panels: Immunofluorescence (IF) for CD81 and HA‐tag in non‐permeabilized HeLa cells, Left two panels: IF for CD81 and HA‐tag in permeabilized HeLa cells. Counterstaining with Hoechst 33342 for DNA. (e) Western Blot of HeLa cells expressing CD81‐snorkel‐tag treated with PreScission protease for indicated time periods mentioned above. The blots were probed with antibodies against HA‐tag and CLIP‐tag. The mean intensity of the bands was quantified and the ratio of the HA‐tag versus CLIP‐tag is shown.

Article Snippet: For quantification of cellular uptake of StEVAC purified EVs, equal number of eluted EVs from CD81‐Snorkel‐tag, WT and CD81‐Snorkel‐tag were labelled with CLIP‐substrate (CLIP‐Surface™ 647; Catalogue #S9234, NEB and/or CLIP‐Surface™ 488; Catalogue #S9232, NEB) with a final dilution of 1:100,000 was added and incubated for 1 h at 95% humidity, 5% CO 2 and 37°C.

Techniques: Expressing, Staining, Membrane, Western Blot, FLAG-tag, Control, Immunofluorescence

Journal: Journal of Extracellular Vesicles

Article Title: Snorkel‐tag based affinity chromatography for recombinant extracellular vesicle purification

doi: 10.1002/jev2.12523

Figure Lengend Snippet: Snorkel‐tagged EVs isolated by tangential flow filtration (TFF) show similar characteristics as wild type EVs. (a) and (b) NTA analysis of TFF isolated EVs from HeLa‐WT and HeLa‐CD81‐snorkel‐tag for particle concentration and size. (c) Western blot of HeLa‐WT and HeLa‐CD81‐Snorkel‐tag cell lysates and EV lysates for Snorkel‐tag epitopes and EV specific markers. (d) Transmission electron microscopy (TEM) images for HeLa‐WT EVs and HeLa‐CD81‐Snorkel‐tag carrying EVs. Overview image (Left, scale bar 1 µm) and close‐up (right, scale bar 200 nm). White arrows label CD81 (anti‐mouse antibody tagged to10 nm gold particle) and red arrows label Snorkel‐tag (anti‐rabbit antibody tagged to 4 nm gold particle) (scale bar: 100 nm). (e) Multiplex bead‐based flow cytometry assay for detection of EV surface protein signature by pan tetraspanin detection antibodies and anti‐HA antibody.

Article Snippet: For quantification of cellular uptake of StEVAC purified EVs, equal number of eluted EVs from CD81‐Snorkel‐tag, WT and CD81‐Snorkel‐tag were labelled with CLIP‐substrate (CLIP‐Surface™ 647; Catalogue #S9234, NEB and/or CLIP‐Surface™ 488; Catalogue #S9232, NEB) with a final dilution of 1:100,000 was added and incubated for 1 h at 95% humidity, 5% CO 2 and 37°C.

Techniques: Isolation, Filtration, Concentration Assay, Western Blot, Transmission Assay, Electron Microscopy, Multiplex Assay, Flow Cytometry

Journal: Journal of Extracellular Vesicles

Article Title: Snorkel‐tag based affinity chromatography for recombinant extracellular vesicle purification

doi: 10.1002/jev2.12523

Figure Lengend Snippet: Snorkel‐tag based extracellular vesicle affinity purification. (a) Workflow for StEVAC method. Created with BioRender.com. (b) Nanoparticle tracking analysis (NTA) counts and (c) particle diameter of purified EVs eluted after incubation with PreScission protease overnight and a following wash step ( n = 7). (d) Transmission electron microscopic examination for size and morphology of the input and eluted EVs (left panel with overview image, scale bar 1 µm; right panel with close‐up image, scale bar 200 nm). (e) Multiplex bead‐based flow cytometry assay to evaluate the StEVAC method. Assay results for HeLa‐WT (top) and HeLa‐CD81‐Snorkel‐tag (bottom); input, flow through (unbound EVs) and elution ( n = 3). (f) Western blots for the EV‐associated protein syntenin and non‐EV marker calnexin (ER specific) in the eluates. One‐way ANOVA was applied on raw values; ns p > 0.05, ** p < 0.01, *** p < 0.001.

Article Snippet: For quantification of cellular uptake of StEVAC purified EVs, equal number of eluted EVs from CD81‐Snorkel‐tag, WT and CD81‐Snorkel‐tag were labelled with CLIP‐substrate (CLIP‐Surface™ 647; Catalogue #S9234, NEB and/or CLIP‐Surface™ 488; Catalogue #S9232, NEB) with a final dilution of 1:100,000 was added and incubated for 1 h at 95% humidity, 5% CO 2 and 37°C.

Techniques: Affinity Purification, Purification, Incubation, Transmission Assay, Multiplex Assay, Flow Cytometry, Western Blot, Marker

Journal: Journal of Extracellular Vesicles

Article Title: Snorkel‐tag based affinity chromatography for recombinant extracellular vesicle purification

doi: 10.1002/jev2.12523

Figure Lengend Snippet: StEVAC specifically isolates snorkel‐tagged EVs out of complex mixtures. (a) Pre‐binding of anti‐HA matrix with HA peptide shows no binding of CD81‐Snorkel‐tag EVs henceforth no EVs in elution. Nanoparticle analysis (NTA) shows significant differences in elutions between anti‐HA matrix pre‐bound with HA peptide and free paratope ( n = 3). (b) Nanoparticle tracking analysis (NTA) counts of purified EVs from HeLa‐CD81‐Snorkel‐tag, HDF76 and mix of both, eluted after incubation with PreScission protease treatment overnight and a following wash step ( n = 3). No significant differences were observed in size distribution between flow through, washes and elution compared to input between CD81‐Snorkel‐tag EVs alone or mixed HDF76 EVs. (c) Multiplex bead‐based assay results for input, flow through and elution of EVs from HeLa‐CD81‐Snorkel‐tag, HDF76 and HeLa‐ CD81‐Snorkel‐tag mixed with HDF76. CD24, CD146 and HLA‐ABC are enriched only in the eluates of HeLa‐CD81‐Snorkel‐tag EVs (c; top row), additionally, tetraspanins CD9, CD63 & CD81 are enriched in all the elutes except in HDF76 (C; bottom row). Particle size quantification revealed no significant size difference during the isolation process ( n = 3). One‐way ANOVA multiple comparison test was used for analysis of (a); 2‐way ANOVA multiple comparison test was used for analysis of (b) and (c). Data are shown as mean+_SD, * p < 0.05, ** p < 0.01, *** p < 0.001.

Article Snippet: For quantification of cellular uptake of StEVAC purified EVs, equal number of eluted EVs from CD81‐Snorkel‐tag, WT and CD81‐Snorkel‐tag were labelled with CLIP‐substrate (CLIP‐Surface™ 647; Catalogue #S9234, NEB and/or CLIP‐Surface™ 488; Catalogue #S9232, NEB) with a final dilution of 1:100,000 was added and incubated for 1 h at 95% humidity, 5% CO 2 and 37°C.

Techniques: Binding Assay, Purification, Incubation, Multiplex Assay, Bead-based Assay, Isolation, Comparison

Journal: Journal of Extracellular Vesicles

Article Title: Snorkel‐tag based affinity chromatography for recombinant extracellular vesicle purification

doi: 10.1002/jev2.12523

Figure Lengend Snippet: Confirming StEVAC for purification of EVs from complex matrices. StEVAC purification and characterization of HeLa‐CD81 Snorkel‐tag EVs from human platelet EV enriched mix. (a) Nanoparticle tracking analysis (NTA) counts of purified EVs from HeLa‐CD81‐Snorkel‐tag and mix of human platelet EVs ( n = 3 individual donors), eluted after incubation with PreScission protease treatment overnight and a following wash step. (b) Multiplex bead‐based flow cytometry (MBFCM) assay results for input, flow through and elution of EVs from HeLa‐CD81‐Snorkel‐tag and HeLa‐ CD81‐Snorkel‐tag mixed with human‐platelet EVs. CD44, CD146 and MCSP1 are enriched only in the elutes for HeLa‐CD81‐Snorkel‐tag EVs (right column), platelet EV specific CD31, CD41b, CD42a, CD62P, CD40 and CD49e are depleted (centre 2 columns). StEVAC purification and characterization of HeLa‐CD81 Snorkel‐tag EVs from human plasma mix. (c) Nanoparticle tracking analysis (NTA) counts of purified EVs from HeLa‐CD81‐Snorkel‐tag and mix of human platelet EVs, eluted after incubation with PreScission protease treatment overnight and a following wash step ( n = 3). (d) Multiplex bead‐based flow cytometry (MBFCM) assay results for input, flow through and elution of EVs from HeLa‐CD81‐Snorkel‐tag and HeLa‐ CD81‐Snorkel‐tag mixed with human‐plasma EVs. CD44, CD146 and MCSP1 are enriched only in the elutes for HeLa‐CD81‐Snorkel‐tag EVs (right column), plasma EV specific CD31, CD41b, CD42a, CD62P, CD69 and HLA‐DRDPDQ are de‐riched (centre 2 columns). Tetraspanins CD9, CD63 and CD81 are enriched equally in both the elutes. (e) Flowcytometric analysis of uptake StEVAC purified EVs from mixture of platelet concentrates labelled with CLIP‐505 substrate into Huh‐7 cells. Unpurified EVs (or) input EVs and flow through EVs were used as control ( n = 3). Same number of particles were used for controls and elutes in uptake. One‐way ANOVA was applied on values; ns p > 0.05, * p < 0.05, ** p < 0.01, *** p < 0.001. 2‐way ANOVA multiple comparison test was used for analysis of (a) and (c). One‐way ANOVA multiple comparison test was used for analysis of (b), (d) and (e). Data are shown as mean + SD, * p < 0.05, ** p < 0.01, *** p < 0.001.

Article Snippet: For quantification of cellular uptake of StEVAC purified EVs, equal number of eluted EVs from CD81‐Snorkel‐tag, WT and CD81‐Snorkel‐tag were labelled with CLIP‐substrate (CLIP‐Surface™ 647; Catalogue #S9234, NEB and/or CLIP‐Surface™ 488; Catalogue #S9232, NEB) with a final dilution of 1:100,000 was added and incubated for 1 h at 95% humidity, 5% CO 2 and 37°C.

Techniques: Purification, Incubation, Multiplex Assay, Flow Cytometry, Control, Comparison

Journal: Journal of Extracellular Vesicles

Article Title: Snorkel‐tag based affinity chromatography for recombinant extracellular vesicle purification

doi: 10.1002/jev2.12523

Figure Lengend Snippet: Influence of Snorkel‐tag on EV cargo loading. StEVAC purification of EVs from WT and CD81‐Snorkel‐tag WJ‐MSCs. (a) Nanoparticle analysis (NTA) shows significant differences in elutaes of WT versus CD81‐Snorkel‐tag EVs. (b) no significant differences were observed in size distribution between flow through, washes and elution compared to input. (c) Bead‐based flow cytometry evaluation of flow throughs, washes and eluates from WJ‐MSC‐WT and WJ‐MSC‐CD81‐Snorkel‐tag EVs for CD63, FLAG‐tag and CD9. (d) Pearson correlation scatter plots of miRNAs expression levels between WJ‐MSC derived EVs isolated by anti‐CD81 pull‐down from CD81‐Snorkel‐tag enriched EVs and WT EVs ( n = 3). (e) Pearson correlation scatter plots of miRNAs expression levels between WJ‐MSC derived CD81‐Snorkel‐tag enriched EVs purified by either CD81 specific pull‐down or by Snorkel‐tag pull down shows only minor differences in cargo loading ( n = 3).

Article Snippet: For quantification of cellular uptake of StEVAC purified EVs, equal number of eluted EVs from CD81‐Snorkel‐tag, WT and CD81‐Snorkel‐tag were labelled with CLIP‐substrate (CLIP‐Surface™ 647; Catalogue #S9234, NEB and/or CLIP‐Surface™ 488; Catalogue #S9232, NEB) with a final dilution of 1:100,000 was added and incubated for 1 h at 95% humidity, 5% CO 2 and 37°C.

Techniques: Purification, Flow Cytometry, FLAG-tag, Expressing, Derivative Assay, Isolation

Journal: Journal of Extracellular Vesicles

Article Title: Snorkel‐tag based affinity chromatography for recombinant extracellular vesicle purification

doi: 10.1002/jev2.12523

Figure Lengend Snippet: The StEVAC platform. Overview on Snorkel‐tag based EV affinity chromatography (StEVAC): (1) CD81‐Snorkel‐tag enriched EVs from ex vivo or in vivo , incubated overnight with anti‐HA matrix at 4°C to capture Snorkel‐tag enriched EVs. (2) Mild treatment of captured EVs with PreScission protease at 4°C releases EVs without changing their biophysical properties. (3) StEVAC method enables to understand the cargo of EVs under normal and disease physiological states, when expressed under tissue‐specific promoter in transgeneic mouse models. Created with BioRender.com.

Article Snippet: For quantification of cellular uptake of StEVAC purified EVs, equal number of eluted EVs from CD81‐Snorkel‐tag, WT and CD81‐Snorkel‐tag were labelled with CLIP‐substrate (CLIP‐Surface™ 647; Catalogue #S9234, NEB and/or CLIP‐Surface™ 488; Catalogue #S9232, NEB) with a final dilution of 1:100,000 was added and incubated for 1 h at 95% humidity, 5% CO 2 and 37°C.

Techniques: Affinity Chromatography, Ex Vivo, In Vivo, Incubation