Journal: Journal of Extracellular Vesicles

Article Title: Signal Amplification for Fluorescent Staining of Single Particles in Liquid Biopsies: Circulating Tumour Cells and Extracellular Vesicles

doi: 10.1002/jev2.70167

Figure Lengend Snippet: (A) Characterization of the size distribution of the EVs isolated from GBM2 CTCs by Izon SEC using NTA. Measured total concentration: 0.9 × 10 9 particles/mL. (B) Characterization of the GBM2 EV protein content (CD9 and EGFR) using ELISA. (C) Characterization of the EV tetraspanin expression and distribution using ONI dSTORM imaging system. CD9‐AF488 (yellow dots), CD63‐AF555 (blue dots) and CD81‐AF594 (pink dots) targeted and analysed. Right hand‐side image: zoom‐in on two EVs co‐expressing the three analysed tetraspanins. The numbers reported beside each tetraspanin represent the number of dots detected on each vesicle and are proportional to the amount/expression of that specific tetraspanin on the EV surface. (D) Validation of the EV capture onto a glass substrate. EVs labelled with tdTomato fluorophore were adsorbed overnight on a TB380 glass slide and imaged the day after, after several washing steps. Two different concentrations (Low EVs = [1×] = 3 × 10 7 particles/mL and High EVs = [8×] = 24 × 10 7 particles/mL) tested, and PSB used as a control. EV counts obtained from four FOVs and plotted on the right as mean ± SD. Images obtained with the 100× oil immersion lens (NA 1.45) of a widefield microscope (Nikon 90i) equipped with a cooled CCD camera (Andor Clara DR‐2519) and a 1.6× optical coupler (Nikon Digital Imaging Head).

Article Snippet: The membranes were incubated overnight with primary antibodies (1:500 dilutions in 5% BSA with 0.02% sodium azide prepared in 1× TBST): HSP70 (cat. # 4872, Cell Signalling), CD63 (cat. # NB100‐77913, Novus Biologicals) and CD9 (cat. # 312102, BioLegend).

Techniques: Isolation, Concentration Assay, Enzyme-linked Immunosorbent Assay, Expressing, Imaging, Biomarker Discovery, Control, Microscopy

Journal: Journal of Extracellular Vesicles

Article Title: Signal Amplification for Fluorescent Staining of Single Particles in Liquid Biopsies: Circulating Tumour Cells and Extracellular Vesicles

doi: 10.1002/jev2.70167

Figure Lengend Snippet: Comparison between DS, PSS and the developed TSA staining protocol using GBM2 EVs. (A, D, G) Representative images of the EVs stained using the three different techniques along with the respective control substrates (no EVs, PBS only + CD63 antibodies). (B, E, H) Fluorescent intensities (plotted as integrated intensities over all the pixels of each single EV) and counts of single EVs stained with the three different techniques, calculated considering two FOVs per technique. (C, F, I) Profiles of the EV pixel intensities over a 5‐min period of continuous laser excitation for the three analysed techniques. The four curves correspond to the pixel intensity distributions of the snapshot images captured at time 0, and after 1, 3 and 5 min of laser excitations, respectively.

Article Snippet: The membranes were incubated overnight with primary antibodies (1:500 dilutions in 5% BSA with 0.02% sodium azide prepared in 1× TBST): HSP70 (cat. # 4872, Cell Signalling), CD63 (cat. # NB100‐77913, Novus Biologicals) and CD9 (cat. # 312102, BioLegend).

Techniques: Comparison, Staining, Control

Journal: Scientific Reports

Article Title: Different populations of Wnt-containing vesicles are individually released from polarized epithelial cells

doi: 10.1038/srep35562

Figure Lengend Snippet: ( a ) Western blot analysis to examine relative amounts of exosome marker proteins, CD63, CD81, Tsg101, and Hsp70, recovered in P100 pellets of apical or basolateral medium of Wnt3a-expressing MDCK (MDCK-3a) cells. Equal amounts of P100 samples prepared from apical or basolateral medium were subjected to Western blotting. As standards, small amounts of cell lysates were also loaded. ( b ) The amounts of Wnt3a in S10 sup and P100 pellet from apical or basolateral medium of MDCK-3a cells, as well as the amount in the cell lysate, were analyzed by Western blotting. To compare the amounts of Wnt3a proteins between apical and basolateral media, an equal volume of S10 (10 μl each for apical or basolateral S10 pool) or equal amount of P100 (1/40 of P100 pellets prepared from 400 ml of apical or basolateral media) samples was loaded. In all MDCK cultures in this study, the same volume of medium was added to both apical and basolateral chambers. ( c–f ) Western blotting analysis for detection of activity and amount of Wnt proteins recovered in S10 sup, S100 sup or P100 pellet prepared from either the apical ( c ) or basolateral side ( e ) of MDCK cells. For monitoring of the Wnt3a activity, the amount of stabilized β-catenin induced by the addition of S10 sup, S100 sup or P100 pellet was quantified in L cells, in which β-catenin is undetectable without the addition of Wnt proteins (control; c,e ). In parallel, the amount of Wnt3a used in this activity assay was analyzed ( c,e ). The amounts of β-catenin and Wnt3a in S10, S100 and P100, shown in ( c ) or ( e ), were quantified using Image J software. The ratios of β-catenin to Wnt3a level are indicated in numerical values relative to that in S10, which was set as 100 ( d,f ). The results shown are the mean ± S.D. from 3 independent experiments. Full-length blots are presented in .

Article Snippet: The P100 pellet isolated from apical MDCK conditioned medium was incubated with 1 × 10 5 anti-CD63 or isotype control conjugated beads in 300 μL wash buffer (PBS containing 2% exosome-depleted FCS) overnight at 4 °C under gentle agitation.

Techniques: Western Blot, Marker, Expressing, Activity Assay, Software

Journal: Scientific Reports

Article Title: Different populations of Wnt-containing vesicles are individually released from polarized epithelial cells

doi: 10.1038/srep35562

Figure Lengend Snippet: ( a,b ) The P100 pellet of either apical ( a ) or basolateral ( b ) conditioned medium from Wnt3a-expressing MDCK (MDCK-3a) cells was subjected to 0.25–2 M continuous sucrose density-gradient centrifugation. Equal volumes of the collected fractions were analyzed by Western blotting to detect Wnt3a and several markers of exosomes, including Flotillin2 (Flo2), CD63, and Tsg101. In addition, contamination of the ER in the apical sample was examined by use of Calreticulin antibody. Distribution of Hsp70 in apical P100 fractionations is indicated separately in comparison with that of CD63. The results are representative of 4 independent experiments. ( c ) Immunoprecipitation analysis to detect Wnt3a in CD63-containing vesicles. The P100 pellet prepared from apically secreted conditioned medium was incubated with anti-CD63-beads or IgG isotype-beads, and the complexes on the beads were recovered and examined with anti- Wnt3a and CD63 antibodies. ( d ) Immuno-electron microscopy of Wnt3a-containing exosomes released into apical and basolateral media. Vesicles stained with anti-Wnt3a antibody and gold particle-conjugated second antibody were observed by transmission electron microscopy. Full-length blots are presented in .

Article Snippet: The P100 pellet isolated from apical MDCK conditioned medium was incubated with 1 × 10 5 anti-CD63 or isotype control conjugated beads in 300 μL wash buffer (PBS containing 2% exosome-depleted FCS) overnight at 4 °C under gentle agitation.

Techniques: Expressing, Gradient Centrifugation, Western Blot, Comparison, Immunoprecipitation, Incubation, Immuno-Electron Microscopy, Staining, Transmission Assay, Electron Microscopy

Journal: Scientific Reports

Article Title: Different populations of Wnt-containing vesicles are individually released from polarized epithelial cells

doi: 10.1038/srep35562

Figure Lengend Snippet: ( a ) Detection of Wnt11 (indicated by the closed arrow) in culture supernatant (Culture Sup.) and in P100 pellets. MDCK cells expressing Wnt11 were cultured in transwells, and soluble Wnt11 in the culture medium was concentrated by use of Blue Sepharose. Then, concentrated samples were suspended in distilled water; and P100 pellets were prepared as described for Wnt3a. Arrowheads indicate the position of Wnt11 protein. Upper bands shown in culture sup. are non-specifically cross-reactive. ( b ) Sucrose density-gradient centrifugation analysis of Wnt11. The P100 pellet prepared from the apical side of Wnt11-expressing MDCK cells was further fractionated by sucrose density-gradient as was shown in . Wnt11, Flo2, and CD63 were analyzed by Western blotting. (Full-length and smaller forms of CD63 are indicated by the closed and open arrow, respectively) Results shown are representative of 2 independent experiments. Full-length blots are presented in .

Article Snippet: The P100 pellet isolated from apical MDCK conditioned medium was incubated with 1 × 10 5 anti-CD63 or isotype control conjugated beads in 300 μL wash buffer (PBS containing 2% exosome-depleted FCS) overnight at 4 °C under gentle agitation.

Techniques: Expressing, Cell Culture, Gradient Centrifugation, Western Blot

Journal: Scientific Reports

Article Title: Different populations of Wnt-containing vesicles are individually released from polarized epithelial cells

doi: 10.1038/srep35562

Figure Lengend Snippet: ( a ) Western blot analysis of P100 pellets to detect recoveries of Wnt3a mutated at the lipidation site, Ser209, produced by MDCK cells. The amounts of Wnt3a in S10 sup and P100 pellet from conditioned medium, as well as the amount in the cell lysate, from MDCK cells expressing the lipidation site mutant Wnt3a (S209A), in which Ser209 was substituted to Ala, were analyzed by Western blotting. Comparative analysis with samples prepared from wild-type Wnt3a-expressing MDCK cells is shown in . ( b,c ) Sucrose density-gradient centrifugation analysis of the lipidation site mutant. The P100 pellet from either the apical ( b ) or basolateral ( c ) side of MDCK cells expressing the S209A mutant of Wnt3a was subjected to continuous sucrose density-gradient as in . The amounts of Wnt3a, Flotillin2 (Flo2), CD63, and Tsg101 were analyzed by Western blotting. Results shown are representative of 3 independent experiments. Full-length blots are presented in .

Article Snippet: The P100 pellet isolated from apical MDCK conditioned medium was incubated with 1 × 10 5 anti-CD63 or isotype control conjugated beads in 300 μL wash buffer (PBS containing 2% exosome-depleted FCS) overnight at 4 °C under gentle agitation.

Techniques: Western Blot, Produced, Expressing, Mutagenesis, Gradient Centrifugation