Journal: Molecular & Cellular Proteomics : MCP

Article Title: The Proteome of Large or Small Extracellular Vesicles in Pig Seminal Plasma Differs, Defining Sources and Biological Functions

doi: 10.1016/j.mcpro.2023.100514

Figure Lengend Snippet: Characterization of extracellular vesicle (EVs) subsets, namely, small (S-EVs) or large (L-EVs), isolated from pig seminal plasma (SP) samples (n = 3; three ejaculates per sample; one ejaculate per male pig). A , violin plot displaying the total protein concentration in both SP-EV subsets. The dashed line shows the median and dotted lines the 25 to 75% quartiles. B , representative histogram of particle size distribution of S-EVs and L-EVs assessed by nanoparticle tracking analysis. C , particle size distribution of S-EVs and L-EVs analyzed by dynamic light scattering ( red , S-EVs; blue , L-EVs) in terms of intensity and volume. The black and gray lines represent the average of intensity size distribution of S-EVs and L-EVs, respectively. D , representative images of the morphology of S-EVs and L-EVs assessed by transmission electron microscopy. E , representative histogram of CFSE/CD63/HSP90β/ALB expression in S-EVs and L-EVs assessed by flow cytometry. ALB, albumin; CFSE, carboxyfluorescein succinimidyl ester; CNT, control; HSP90β, heat shock protein 90β.

Article Snippet: The EVs were cytometrically characterized following the International Society of Extracellular Vesicles recommendations (MIFlowCyt-EV, ( )) to identify their enrichment in proteins belonging to the three categories established by MISEV 2018 guidelines ( ): CD63 (Anti-CD63-FITC, Clone REA1055, Miltenyi Biotec) as “Category 1” protein (Transmembrane or GPI-anchored proteins associated to plasma membrane and/or endosomes); HSP90β (anti-HSP90β-PE, ADI-SPA-844PE-050, Enzo Life Sciences) as “Category 2” protein (Cytosolic proteins recovered in EVs), and albumin (Anti-swine Albumin-FITC, CLFAG16140, Cedarlane) as “Category 3” protein (Major components of non-EVs coisolated structures).

Techniques: Isolation, Clinical Proteomics, Protein Concentration, Transmission Assay, Electron Microscopy, Expressing, Flow Cytometry, Control

Journal: Scientific Reports

Article Title: Extracellular vesicles cargo from head and neck cancer cell lines disrupt dendritic cells function and match plasma microRNAs

doi: 10.1038/s41598-021-97753-y

Figure Lengend Snippet: EVs characterization by size, imaging and tetraspanin expression. ( A ) Size distribution of EVs in SCC25 and FaDu culture medium. EV sizes were plotted over calibration particles diameters; ( B ) Representative images of transmission electron microscopy of EVs isolated from cell culture medium at × 2100 and × 3500 magnification, the selected images were obtained from FaDu culture medium but identical results were obtained for SCC25; ( C ) First graph displays the percentage of tetraspanin expression (CD9, CD63 and CD81) by EVs derived from FaDu cell lines and second graph displays the percentage of tetraspanin expression (CD9, CD63 and CD81) by EVs derived from SSC25 cell lines; ( D ) Flow cytometry control of fluorescence and size determination using Megamix-Plus FSC fluorescent particles with different sizes (0.1–0.9 µM); ( E ) Representative dot plot graphs displaying fow cytometry analysis of tetraspanin (CD9, CD63 and CD81) expression by EVs derived from FaDu cell line; ( F ) Representative dot plot graphs displaying fow cytometry analysis of tetraspanin expression by EVs derived from SCC25 cell line.

Article Snippet: Immunophenotyping was performed for EVs following current protocols using the following antibodies: CD9-PE (clone:209306) from R&D systems, CD63-FITC (clone: MEM-259) from USBiological and CD81-FITC (clone:JS64) from Beckman Coulter.

Techniques: Imaging, Expressing, Transmission Assay, Electron Microscopy, Isolation, Cell Culture, Derivative Assay, Flow Cytometry, Control, Fluorescence, Cytometry

Journal: International Journal of Molecular Sciences

Article Title: Basophil Activation Test with Different Polyethylene Glycols in Patients with Suspected PEG Hypersensitivity Reactions

doi: 10.3390/ijms232314592

Figure Lengend Snippet: Gating strategy. SSClow/CCR3+ events were firstly selected on an SSC-A/CCR3-PE dot-plot, and then CD63-positive basophils were identified on a CCR3-PE/CD63-FITC dot-plot for the negative control (C−), positive controls (C+ FcεRI and C+ fMLP) and allergens (PEG 2000, PEG 4000 and DMG-PEG 2000). The gating strategy is representative of all analyzed samples.

Article Snippet: The samples were stained using an anti-CCR3-Phycoerythrin (PE)-conjugated antibody and an anti-CD63- fluorescein isothiocyanate (FITC)-conjugated antibody (Flow CAST ® kit, Buhlmann Laboratories, Schonenbuch, Switzerland) and incubated for 15 min at 37 °C in a water bath after which the samples underwent a red blood cell lysis step (10 min, RT); finally, the samples were centrifuged for 10 min at 400× g and resuspended by adding 300 µL of wash buffer before the acquisition.

Techniques: Negative Control

Journal: International Journal of Molecular Sciences

Article Title: Basophil Activation Test with Different Polyethylene Glycols in Patients with Suspected PEG Hypersensitivity Reactions

doi: 10.3390/ijms232314592

Figure Lengend Snippet: Stimulation index (SI) of CD63 expression. The graphs show the SI of the control group, patients that were negative for the test (BAT−), and patients that were considered positive (BAT+). SI > 2.5 was considered as positivity for the test.

Article Snippet: The samples were stained using an anti-CCR3-Phycoerythrin (PE)-conjugated antibody and an anti-CD63- fluorescein isothiocyanate (FITC)-conjugated antibody (Flow CAST ® kit, Buhlmann Laboratories, Schonenbuch, Switzerland) and incubated for 15 min at 37 °C in a water bath after which the samples underwent a red blood cell lysis step (10 min, RT); finally, the samples were centrifuged for 10 min at 400× g and resuspended by adding 300 µL of wash buffer before the acquisition.

Techniques: Expressing

Journal: Journal of Extracellular Vesicles

Article Title: An aptamer‐guided fluorescence polarisation platform for extracellular vesicle liquid biopsy

doi: 10.1002/jev2.12502

Figure Lengend Snippet: Sequences of aptamers used in this study.

Article Snippet: After that, 100 μL of 5.0 nM FITC‐conjugated CD63 aptamer (CD63‐BP) or FITC‐conjugated HER2 aptamer (HER2‐HApt) was introduced and incubated with the well for 1 h in the dark on a shaker (Thermoline Scientific, Model No.: TL400) at 120 rpm at room temperature.

Techniques: Biomarker Discovery, Control, Clinical Proteomics, Membrane

Journal: Journal of Extracellular Vesicles

Article Title: An aptamer‐guided fluorescence polarisation platform for extracellular vesicle liquid biopsy

doi: 10.1002/jev2.12502

Figure Lengend Snippet: Optimisations of the FluoPADE assays. (a‒e), Optimisation of EV isolation. (a) Schematic illustration of EV immobilisation using antibodies followed by the detection using FITC‐conjugated anti‐CD63 antibody. (b) HT‐29 EVs were captured using biotinylated anti‐EpCAM antibody, EVs from SKRB3 cells were captured using 1:1 molar ratio of biotinylated anti‐CD9/CD81 antibodies. As a control of particles versus vesicles, the captured EVs were treated with 1% Triton X‐100 or saline prior to immobilisation. **** p < 0.0001 compared with EVs treated with Triton X‐100. (c) Optimisation of concentration of biotinylated anti‐EpCAM antibody for HT‐29 EV capture and that of biotinylated anti‐CD9/CD81 antibodies for capturing EVs from SKBR3 cells. * p < 0.05 compared with fluorescence intensity for EVs immobilised using 5 µg/mL antibody. (d) Optimisation of well‐coating time for biotinylated anti‐EpCAM antibody for HT‐29 EV capture and that for the mixture of anti‐CD9/CD81 antibodies for SKBR3 EV capture. (e) Optimisation of capture time for HT‐29 EVs by anti‐EpCAM antibody or SKBR3 EVs by anti‐CD9/CD81antibodies. * p < 0.05 compared with fluorescence intensity of the group of 4 h incubation. (f‒i) Optimisation of aptamer concentration. (f) Signal‐to‐noise ratios of parallel and perpendicular fluorescence intensity for free FITC‐labelled CD63‐BP aptamer at various concentrations over that of EV sample without aptamers. (g) FP of free FITC‐labelled CD63‐BP aptamer at the concentrations indicated in the absence of EVs. (h) Signal‐to‐noise ratios of parallel and perpendicular intensity for free FITC‐labelled HER2‐HApt aptamer at the concentrations indicated over that of EV sample without aptamers. (i) FP of FITC‐labelled HER2‐HApt aptamer at the concentrations indicated in the absence of EVs. (j and k) Optimisation of incubation time for aptamers and immobilised EV. (j) The fluorescence polarisation of FITC‐CD63‐BP aptamer added to the immobilised HT‐29 EVs. (k) The fluorescence polarisation of FITC‐HER2‐HApt aptamer added to the immobilised SKRB3 EVs. * p < 0.05; ns: not significant. Data shown are means ± S.D, n = 3.

Article Snippet: After that, 100 μL of 5.0 nM FITC‐conjugated CD63 aptamer (CD63‐BP) or FITC‐conjugated HER2 aptamer (HER2‐HApt) was introduced and incubated with the well for 1 h in the dark on a shaker (Thermoline Scientific, Model No.: TL400) at 120 rpm at room temperature.

Techniques: Isolation, Control, Saline, Concentration Assay, Fluorescence, Incubation

Journal: Journal of Extracellular Vesicles

Article Title: An aptamer‐guided fluorescence polarisation platform for extracellular vesicle liquid biopsy

doi: 10.1002/jev2.12502

Figure Lengend Snippet: Specificity of FluoPADE assays. (a) Difference in fluorescence polarisation of 5 nM FITC‐CD63‐BP aptamer at a detection ligand against six different analytes: EVs from HT‐29 cells immobilised using anti‐EpCAM antibody; FITC‐negative control aptamer incubated with HT‐29 EVs immobilised using anti‐EpCAM antibody; HT‐29 EVs immobilised using anti‐EpCAM antibody followed by the lysis using 1% Triton X‐100; HT‐29 EVs incubated with the microwell coated with IgG isotype control antibody; EVs from HEK293 cells that do not express EpCAM; and immobilised EpCAM protein without the input EVs. (b) Difference in fluorescence polarisation of 5 nM FITC‐HER2‐HApt aptamer as a detection ligand against five different analytes: SKRB3 EVs immobilised using anti‐CD9/CD81 antibodies; FITC‐negative control aptamer incubated with SKRB3 EVs immobilised using anti‐CD9/CD81 antibodies; SKRB3 EVs immobilised using anti‐CD9/CD81 antibodies followed by the lysis with 1% Triton X‐100; SKRB3 EVs incubated with the microwell coated with isotype control antibody; HER2‐negative EVs from MDA‐MB‐231cells with the HER2 gene knocked out. Data shown as means ± S.D., n = 3.

Article Snippet: After that, 100 μL of 5.0 nM FITC‐conjugated CD63 aptamer (CD63‐BP) or FITC‐conjugated HER2 aptamer (HER2‐HApt) was introduced and incubated with the well for 1 h in the dark on a shaker (Thermoline Scientific, Model No.: TL400) at 120 rpm at room temperature.

Techniques: Fluorescence, Negative Control, Incubation, Lysis, Control

Journal: Journal of Extracellular Vesicles

Article Title: An aptamer‐guided fluorescence polarisation platform for extracellular vesicle liquid biopsy

doi: 10.1002/jev2.12502

Figure Lengend Snippet: Limit of detection and linear dynamic range of FluoPADE assays. Changes in the fluorescence polarisation as a function of EV concentration and linear plots of fluorescence polarisation change as a function of the log10 (lg) of EV concentration (inset) are presented, along with limit of detection and linear dynamic range of FluoPADE assays. (a and b) CD63‐BP aptamer with EVs from HT‐29. (c and d) HER2‐HApt aptamer with EVs from SKBR3. Cell line‐derived EVs were diluted at a 1:9 ratio either in PBS (a, c) or in human plasma (b, d). Data shown are means ± S.D., n = 3.

Article Snippet: After that, 100 μL of 5.0 nM FITC‐conjugated CD63 aptamer (CD63‐BP) or FITC‐conjugated HER2 aptamer (HER2‐HApt) was introduced and incubated with the well for 1 h in the dark on a shaker (Thermoline Scientific, Model No.: TL400) at 120 rpm at room temperature.

Techniques: Fluorescence, Concentration Assay, Derivative Assay, Clinical Proteomics

Journal: Journal of Extracellular Vesicles

Article Title: An aptamer‐guided fluorescence polarisation platform for extracellular vesicle liquid biopsy

doi: 10.1002/jev2.12502

Figure Lengend Snippet: Summary of limits of detection (LOD), limit of quantification (LOQ) and linear dynamic ranges of FluoPADE assays and fluorescence intensity (FI) assays.

Article Snippet: After that, 100 μL of 5.0 nM FITC‐conjugated CD63 aptamer (CD63‐BP) or FITC‐conjugated HER2 aptamer (HER2‐HApt) was introduced and incubated with the well for 1 h in the dark on a shaker (Thermoline Scientific, Model No.: TL400) at 120 rpm at room temperature.

Techniques: Fluorescence, Clinical Proteomics

Journal: Journal of Extracellular Vesicles

Article Title: An aptamer‐guided fluorescence polarisation platform for extracellular vesicle liquid biopsy

doi: 10.1002/jev2.12502

Figure Lengend Snippet: The sensitivity of FluoPADE assay in the detection of cancer biomarker‐positive EVs. Changes in fluorescence polarisation of aptamers as a function of decreasing prevalence of EVs that display a cancer biomarker protein on the EV membrane amidst a constant concentration of EV generated by serial dilution of the former with EVs that do not express the cancer biomarker protein. (a) Schematic illustration of the sensitivity assay using FITC‐CD63‐BP aptamer. (b) The ΔFP of serially diluted EpCAM‐positive HT‐29 EVs with EpCAM‐negative HEK293 EVs but maintaining the total EV concentration using FITC‐CD63‐BP aptamer. (c) Schematic illustration of the sensitivity assay using FITC‐HER2‐HApt aptamer. (d) The ΔFP of serially diluted HER2‐positive SKBR3 EVs with HER2‐negative Her2‐gene knockout MDA‐MB‐231 EVs but maintaining the total EV concentration using FITC‐HER2‐HApt aptamer. Data shown are means ± S.D., n = 3.

Article Snippet: After that, 100 μL of 5.0 nM FITC‐conjugated CD63 aptamer (CD63‐BP) or FITC‐conjugated HER2 aptamer (HER2‐HApt) was introduced and incubated with the well for 1 h in the dark on a shaker (Thermoline Scientific, Model No.: TL400) at 120 rpm at room temperature.

Techniques: Biomarker Discovery, Fluorescence, Membrane, Concentration Assay, Generated, Serial Dilution, Sensitive Assay, Gene Knockout

Journal: Journal of Extracellular Vesicles

Article Title: An aptamer‐guided fluorescence polarisation platform for extracellular vesicle liquid biopsy

doi: 10.1002/jev2.12502

Figure Lengend Snippet: The FluoPADE is capable of differentiating EVs displaying the same set of biomarkers but from different sources via multiparametric analysis. (a) Schematic illustrations of FluoPADE assays detecting EVs immobilised using anti‐EpCAM antibody from three different cancer cell lines spiked into human plasma from six individual donors using FITC‐CD63‐BP aptamer. (b) Differential changes in fluorescence polarisation generated by the CD63‐BP aptamer on EVs immobilised with the same antibody to EpCAM from three different cancer cell lines spiked into human plasma from six individual donors. (c) Schematic illustrations of FluoPADE assays detecting EVs immobilised using anti‐CD9/CD81 antibodies from three different cancer cell lines spiked into human plasma from six individual donors with HER2‐HApt aptamer. (d) Differential changes in fluorescence polarisation generated by the HER2‐HApt aptamer on EVs immobilised with the same set of antibodies to CD9/CD81 from three different cancer cell lines spiked into human plasma from six individual donors. (e), Clustering plot of HT‐29 EVs, SKBR3 EVs and HepG2 EVs prepared from ΔFPs using CD63 aptamer as in (b) and ΔFPs using HER2 aptamer as in (d). Data shown are representative of three independent experiments. In all samples, the total EV concentration was maintained at 1.0 × 10 10 EVs/mL; whilst spiked cancer cell line‐derived EVs were kept at 1.0 × 10 9 EVs/mL.

Article Snippet: After that, 100 μL of 5.0 nM FITC‐conjugated CD63 aptamer (CD63‐BP) or FITC‐conjugated HER2 aptamer (HER2‐HApt) was introduced and incubated with the well for 1 h in the dark on a shaker (Thermoline Scientific, Model No.: TL400) at 120 rpm at room temperature.

Techniques: Clinical Proteomics, Fluorescence, Generated, Concentration Assay, Derivative Assay

Journal: Journal of Extracellular Vesicles

Article Title: An aptamer‐guided fluorescence polarisation platform for extracellular vesicle liquid biopsy

doi: 10.1002/jev2.12502

Figure Lengend Snippet: Summary of features of EV detection assays.

Article Snippet: After that, 100 μL of 5.0 nM FITC‐conjugated CD63 aptamer (CD63‐BP) or FITC‐conjugated HER2 aptamer (HER2‐HApt) was introduced and incubated with the well for 1 h in the dark on a shaker (Thermoline Scientific, Model No.: TL400) at 120 rpm at room temperature.

Techniques: Marker, Microscopy, Förster Resonance Energy Transfer, Fluorescence, Colorimetric Assay, SPR Assay, Imaging, Enzyme-linked Immunosorbent Assay