Journal: bioRxiv

Article Title: Evaluation of Tetraspanins in Extracellular Vesicle Bioengineering

doi: 10.64898/2026.01.13.699196

Figure Lengend Snippet: Lentiviral transduction to generate WT-, PanKO-, CD9KO-, CD63KO-, and CD81KO-cells expressing TlucCD9-Cerulean. A. Schematic workflow of generating engineered Tluc-EVs by introducing TlucCD9-Cerulean lentiviruses (created with BioRender.com ). B. The percentage of Cerulean positive cells. C. The mean fluorescence intensity (MFI) of Cerulean positive cells. D. Flow cytometry plot for the cells after staining with APC-conjugated CD9/CD63/CD81 tetraspanin antibodies. E. Fold increase in engineered Tluc-CD9 EVs in PanKO-, CD9KO, CD63KO-, and CD81KO-cells over WT cells (Data are normalized to the RLU of EVs from WT cells). F. Fold increase of engineered Tluc-CD9EVs over PanKO-, CD9KO, CD63KO-, and CD81KO-cells (Data are normalized to the RLU of EVs from WT cells and compared across KO groups). G. Heatmap of tetraspanins in EVs. H. Interaction network of CD9 with the tetraspanins retrieved from STRING. The data are presented as means (±SD, n = 3-5). One-way ANOVA was used to show significance and was illustrated as follows: **** p < 0.0001.

Article Snippet: Briefly, 2.5 × 10 8 EVs, quantified by NTA, were stained with 8 nM fluorescent antibodies targeting the tetraspanins CD9-APC (clone: SN4 C3-3A2, cat. 130-128-037), CD63-APC (clone: H5C6, cat. 130-100-182, Miltenyi Biotec), and CD81-APC (clone: JS64, Beckman Coulter).

Techniques: Transduction, Expressing, Fluorescence, Flow Cytometry, Staining

Journal: bioRxiv

Article Title: Evaluation of Tetraspanins in Extracellular Vesicle Bioengineering

doi: 10.64898/2026.01.13.699196

Figure Lengend Snippet: Lentiviral transduction to generate WT, PanKO, CD9KO, CD63KO, and CD81KO cells expressing TlucCD63-Cerulean. A. Schematic workflow of engineered Tluc-EVs by introducing TlucCD63-Cerulean lentiviruses (created with BioRender.com ). B. The percentage of Cerulean positive cells. C. The mean fluorescent intensity (MFI) of Cerulean positive cells. D. Flow cytometry plot for the cells after staining with APC-conjugated CD9/CD63/CD81 tetraspanin antibodies. E. Fold increase of engineered Tluc-CD63 EVs in PanKO-, CD9KO, CD63KO-, and CD81KO-cells over WT cells (Data are normalized to the RLU of EVs from WT cells). F. Fold increase of engineered Tluc-CD63EVs in between PanKO-, CD9KO, CD63KO-, and CD81KO-cells (Data are normalized to the RLU of EVs from WT cells and compared across KO groups). G. Heatmap of expressing tetraspanins in EVs. H. Interaction network of CD63 with the tetraspanins retrieved from STRING. The data are presented as means (±SD, n = 3-5). One-way ANOVA was used to show significance and was illustrated as follows: * p < 0.05, ** p < 0.01, **** p < 0.0001.

Article Snippet: Briefly, 2.5 × 10 8 EVs, quantified by NTA, were stained with 8 nM fluorescent antibodies targeting the tetraspanins CD9-APC (clone: SN4 C3-3A2, cat. 130-128-037), CD63-APC (clone: H5C6, cat. 130-100-182, Miltenyi Biotec), and CD81-APC (clone: JS64, Beckman Coulter).

Techniques: Transduction, Expressing, Flow Cytometry, Staining

Journal: bioRxiv

Article Title: Evaluation of Tetraspanins in Extracellular Vesicle Bioengineering

doi: 10.64898/2026.01.13.699196

Figure Lengend Snippet: Lentiviral transduction to generate WT-, PanKO-, CD9KO-, CD63KO-, and CD81KO-cells expressing TlucCD9-Cerulean. A. Schematic workflow of engineered Tluc-EVs by introducing TlucCD81-Cerulean lentiviruses (created with BioRender.com ). B. The percentage of Cerulean positive cells. C. The MFI of Cerulean positive cells. D. Flow cytometry plot for the cells after staining with APC-conjugated CD9/CD63/CD81 tetraspanin antibodies. E. Fold increase of engineered Tluc-CD81 EVs in PanKO-, CD9KO, CD63KO-, and CD81KO-cells over WT cells (Data are normalized to the RLU of EVs from WT cells). F. Fold increase of engineered Tluc-CD81EVs in between PanKO-, CD9KO, CD63KO-, and CD81KO-cells (Data are normalized to the RLU of EVs from WT cells and compared across KO groups). G. Heatmap of expressing tetraspanins in EVs. H. Interaction network of CD81 with the tetraspanins retrieved from STRING. The data are presented as means (±SD, n = 3-5). One-way ANOVA was used to show significance and was illustrated as follows: ** p < 0.01,** * p < 0.001,**** p < 0.0001.

Article Snippet: Briefly, 2.5 × 10 8 EVs, quantified by NTA, were stained with 8 nM fluorescent antibodies targeting the tetraspanins CD9-APC (clone: SN4 C3-3A2, cat. 130-128-037), CD63-APC (clone: H5C6, cat. 130-100-182, Miltenyi Biotec), and CD81-APC (clone: JS64, Beckman Coulter).

Techniques: Transduction, Expressing, Flow Cytometry, Staining

Journal: bioRxiv

Article Title: Evaluation of Tetraspanins in Extracellular Vesicle Bioengineering

doi: 10.64898/2026.01.13.699196

Figure Lengend Snippet: Generation of CD63-mNG-EVs in WT, PanKO-, CD9KO-, CD63KO-, and CD81KO-cells. A. Schematic workflow of engineered mNG-EVs by introducing CD63-mNG lentiviruses “created with BioRender.com ”. B. Percentage of mNG positive cells after transduction using flow cytometry. C. MFI of the cells using flow cytometry. D. The flow cytometry plot for the cells after transduction, stained with APC-conjugated CD9/CD63/CD81 tetraspanin antibodies. E. Quantification of engineered CD63-mNG EVs from 17 µL of CM collected from KO) and WT cells. F. Imaging flow cytometry plot for the mNG-EVs derived from stably expressing mNG cells. The data are presented as means (±SD, n = 2-3). One-way ANOVA was used to show significance and was illustrated as follows: * p< 0.05; ** p < 0.01; *** p < 0.001.

Article Snippet: Briefly, 2.5 × 10 8 EVs, quantified by NTA, were stained with 8 nM fluorescent antibodies targeting the tetraspanins CD9-APC (clone: SN4 C3-3A2, cat. 130-128-037), CD63-APC (clone: H5C6, cat. 130-100-182, Miltenyi Biotec), and CD81-APC (clone: JS64, Beckman Coulter).

Techniques: Transduction, Flow Cytometry, Staining, Imaging, Derivative Assay, Stable Transfection, Expressing

Journal: bioRxiv

Article Title: Evaluation of Tetraspanins in Extracellular Vesicle Bioengineering

doi: 10.64898/2026.01.13.699196

Figure Lengend Snippet: Lentiviral transduction to generate WT-, PanKO-, CD9KO-, CD63KO-, and CD81KO-cells expressing TlucCD9-Cerulean. A. Schematic workflow of generating engineered Tluc-EVs by introducing TlucCD9-Cerulean lentiviruses (created with BioRender.com ). B. The percentage of Cerulean positive cells. C. The mean fluorescence intensity (MFI) of Cerulean positive cells. D. Flow cytometry plot for the cells after staining with APC-conjugated CD9/CD63/CD81 tetraspanin antibodies. E. Fold increase in engineered Tluc-CD9 EVs in PanKO-, CD9KO, CD63KO-, and CD81KO-cells over WT cells (Data are normalized to the RLU of EVs from WT cells). F. Fold increase of engineered Tluc-CD9EVs over PanKO-, CD9KO, CD63KO-, and CD81KO-cells (Data are normalized to the RLU of EVs from WT cells and compared across KO groups). G. Heatmap of tetraspanins in EVs. H. Interaction network of CD9 with the tetraspanins retrieved from STRING. The data are presented as means (±SD, n = 3-5). One-way ANOVA was used to show significance and was illustrated as follows: **** p < 0.0001.

Article Snippet: 50 μl of purified EVs at a concentration of 1 × 10 10 /ml were stained with either anti-human CD9 (Miltenyi Biotech, clone SN4), anti-human CD63 (Miltenyi Biotec, clone H5C6) and anti-human CD81 antibodies (Beckman Coulter, clone JS64) or REA control APC conjugated antibodies at a concentration of 8 nM overnight at room temperature in the dark.

Techniques: Transduction, Expressing, Fluorescence, Flow Cytometry, Staining

Journal: bioRxiv

Article Title: Evaluation of Tetraspanins in Extracellular Vesicle Bioengineering

doi: 10.64898/2026.01.13.699196

Figure Lengend Snippet: Lentiviral transduction to generate WT, PanKO, CD9KO, CD63KO, and CD81KO cells expressing TlucCD63-Cerulean. A. Schematic workflow of engineered Tluc-EVs by introducing TlucCD63-Cerulean lentiviruses (created with BioRender.com ). B. The percentage of Cerulean positive cells. C. The mean fluorescent intensity (MFI) of Cerulean positive cells. D. Flow cytometry plot for the cells after staining with APC-conjugated CD9/CD63/CD81 tetraspanin antibodies. E. Fold increase of engineered Tluc-CD63 EVs in PanKO-, CD9KO, CD63KO-, and CD81KO-cells over WT cells (Data are normalized to the RLU of EVs from WT cells). F. Fold increase of engineered Tluc-CD63EVs in between PanKO-, CD9KO, CD63KO-, and CD81KO-cells (Data are normalized to the RLU of EVs from WT cells and compared across KO groups). G. Heatmap of expressing tetraspanins in EVs. H. Interaction network of CD63 with the tetraspanins retrieved from STRING. The data are presented as means (±SD, n = 3-5). One-way ANOVA was used to show significance and was illustrated as follows: * p < 0.05, ** p < 0.01, **** p < 0.0001.

Article Snippet: 50 μl of purified EVs at a concentration of 1 × 10 10 /ml were stained with either anti-human CD9 (Miltenyi Biotech, clone SN4), anti-human CD63 (Miltenyi Biotec, clone H5C6) and anti-human CD81 antibodies (Beckman Coulter, clone JS64) or REA control APC conjugated antibodies at a concentration of 8 nM overnight at room temperature in the dark.

Techniques: Transduction, Expressing, Flow Cytometry, Staining

Journal: bioRxiv

Article Title: Evaluation of Tetraspanins in Extracellular Vesicle Bioengineering

doi: 10.64898/2026.01.13.699196

Figure Lengend Snippet: Lentiviral transduction to generate WT-, PanKO-, CD9KO-, CD63KO-, and CD81KO-cells expressing TlucCD9-Cerulean. A. Schematic workflow of engineered Tluc-EVs by introducing TlucCD81-Cerulean lentiviruses (created with BioRender.com ). B. The percentage of Cerulean positive cells. C. The MFI of Cerulean positive cells. D. Flow cytometry plot for the cells after staining with APC-conjugated CD9/CD63/CD81 tetraspanin antibodies. E. Fold increase of engineered Tluc-CD81 EVs in PanKO-, CD9KO, CD63KO-, and CD81KO-cells over WT cells (Data are normalized to the RLU of EVs from WT cells). F. Fold increase of engineered Tluc-CD81EVs in between PanKO-, CD9KO, CD63KO-, and CD81KO-cells (Data are normalized to the RLU of EVs from WT cells and compared across KO groups). G. Heatmap of expressing tetraspanins in EVs. H. Interaction network of CD81 with the tetraspanins retrieved from STRING. The data are presented as means (±SD, n = 3-5). One-way ANOVA was used to show significance and was illustrated as follows: ** p < 0.01,** * p < 0.001,**** p < 0.0001.

Article Snippet: 50 μl of purified EVs at a concentration of 1 × 10 10 /ml were stained with either anti-human CD9 (Miltenyi Biotech, clone SN4), anti-human CD63 (Miltenyi Biotec, clone H5C6) and anti-human CD81 antibodies (Beckman Coulter, clone JS64) or REA control APC conjugated antibodies at a concentration of 8 nM overnight at room temperature in the dark.

Techniques: Transduction, Expressing, Flow Cytometry, Staining

Journal: bioRxiv

Article Title: Evaluation of Tetraspanins in Extracellular Vesicle Bioengineering

doi: 10.64898/2026.01.13.699196

Figure Lengend Snippet: Generation of CD63-mNG-EVs in WT, PanKO-, CD9KO-, CD63KO-, and CD81KO-cells. A. Schematic workflow of engineered mNG-EVs by introducing CD63-mNG lentiviruses “created with BioRender.com ”. B. Percentage of mNG positive cells after transduction using flow cytometry. C. MFI of the cells using flow cytometry. D. The flow cytometry plot for the cells after transduction, stained with APC-conjugated CD9/CD63/CD81 tetraspanin antibodies. E. Quantification of engineered CD63-mNG EVs from 17 µL of CM collected from KO) and WT cells. F. Imaging flow cytometry plot for the mNG-EVs derived from stably expressing mNG cells. The data are presented as means (±SD, n = 2-3). One-way ANOVA was used to show significance and was illustrated as follows: * p< 0.05; ** p < 0.01; *** p < 0.001.

Article Snippet: 50 μl of purified EVs at a concentration of 1 × 10 10 /ml were stained with either anti-human CD9 (Miltenyi Biotech, clone SN4), anti-human CD63 (Miltenyi Biotec, clone H5C6) and anti-human CD81 antibodies (Beckman Coulter, clone JS64) or REA control APC conjugated antibodies at a concentration of 8 nM overnight at room temperature in the dark.

Techniques: Transduction, Flow Cytometry, Staining, Imaging, Derivative Assay, Stable Transfection, Expressing