Journal: Scientific Reports

Article Title: Co-continuous structural effect of size-controlled macro-porous glass membrane on extracellular vesicle collection for the analysis of miRNA

doi: 10.1038/s41598-021-87986-2

Figure Lengend Snippet: The miRNA array analysis of EVs collected from human serum. ( a ) A schematic diagram of the miRNA array analysis of HepG2-EVs collected from human serum and urine using four EV collection methods (UC, MB, PR, and the AGC device). ( b ) The western blotting of CD9 in serum (upper), and CD9 in the collected and flow fraction of serum treated by AGC device (lower). These blots were cropped from the same gel. The black dot line shows the delineation with dividing from different parts of the same gel. All information about full-length gels and blots was provided in Fig S9. ( c ) The capture rate (%) of EVs using AGC device calculated by CD9/CD63 ELISA kit. ( d ) A gel image of nucleotides [nt] of total RNA extracted from EVs collected from human serum using four EV collection methods (UC, MB, PR, and AGC device) measured by a 2100 Bioanalyzer. ( e ) The concentration of total RNA extracted from EVs collected from human serum using four EV collection methods (UC, MB, PR and the AGC device) measured by a 2100 Bioanalyzer. ( f ) The number of miRNAs extracted from EVs collected from human serum using four EV collection methods (UC, MB, PR, and the AGC device). ( g – i ) A venn diagram of the overlap of the number of detected miRNAs between UC (as the standard method) and the other three methods (MB [ g ], PR [ h ] and the AGC device [ i ]).

Article Snippet: The anti-CD9 (clone 12A12) and anti-CD63 (clone 8A12) antibodies and CD9/CD63 ELISA kit were purchased from Cosmo Bio (Tokyo, Japan).

Techniques: Western Blot, Enzyme-linked Immunosorbent Assay, Concentration Assay

Journal: bioRxiv

Article Title: Salivary exosomal miR-1307-5p predicts disease aggressiveness and poor prognosis in oral squamous cell carcinoma patients

doi: 10.1101/2022.07.13.499918

Figure Lengend Snippet: (a) Representative TEM images of exosomes from OSCC patients, (Scale: 200nm); (b) Estimation of size and concentration of salivary exosomes from OSCC patients using Nanoparticle Tracking Analysis (NTA). The x-axis indicates the size distribution of particles and the y-axis shows the signal intensity in NTA; (c-e) Characterization of exosomes by flow cytometry. We used a membrane-binding dye vFRed to stain exosomes and examined the violet side scatter v/s vFRed staining of these exosomes. This profile was similar to the profile of Lipo-50 liposomes used as a reference standard. Salivary exosomes were stained with antibodies against tetraspanins (CD63, CD81 and CD9) labelled with PE, and APC labelled CD47 antibody. We identified a distinct CD47+/vFRed+ population in the exosome population that was absent in the unstained control. This population was then examined for tetraspanin expression and showed detectable levels of tetraspanins. Since these particles stain with vFRed, have a violet side scatter similar to 50 nm liposomes, express CD47 and tetraspanins, we concluded that these are exosomes.

Article Snippet: The samples (at optimal dilution) were then incubated with PE tagged antibodies against tetraspanins (TS; CD9, CD63, and CD81) (Cellarcus Biosciences, CA, USA) and APC tagged CD47 antibody (Thermo Scientific, Waltham, MA, USA) for one hour at room temperature.

Techniques: Concentration Assay, Flow Cytometry, Membrane, Binding Assay, Staining, Liposomes, Control, Expressing

Journal: Scientific Reports

Article Title: Soluble ectodomain CD163 and extracellular vesicle-associated CD163 are two differently regulated forms of ‘soluble CD163’ in plasma

doi: 10.1038/srep40286

Figure Lengend Snippet: ( A ) Western blot analysis of CD9 and CD163. Blots are cropped for presentation. Full-length blots are available in . ( B ) CD163 ELISA of human plasma before (total plasma/soluble CD163) and after (Non-EV fraction/soluble ectodomain CD163) precipitation of EVs (EV fraction/EV CD163). Data presented as median with interquartile range.

Article Snippet: Immunoblotting was performed using specific antibodies for CD9 and CD163 (pAb rabbit anti-human CD9; Systems Bioscience, mAb rat anti-mouse CD9 (clone MF1); Bio-Rad, UK, mAb mouse anti-human CD163 (clone Mac2–158); IQ products, Netherlands and pAb rabbit anti-mouse CD163) and a secondary HRP conjugated goat anti-rabbit, rat or mouse IgG antibody (Sigma-Aldrich).

Techniques: Western Blot, Enzyme-linked Immunosorbent Assay, Clinical Proteomics

Journal: Scientific Reports

Article Title: Soluble ectodomain CD163 and extracellular vesicle-associated CD163 are two differently regulated forms of ‘soluble CD163’ in plasma

doi: 10.1038/srep40286

Figure Lengend Snippet: ( A ) Beads-based FACS analysis of exosome-FITC and CD9-PE staining on αCD163 (dark grey) or Isotype IgG immuno-precipitated EV’s isolated from human plasma. ( B ) Negative-stain EM of exosomes purified by αCD163 or αCD9 immunoprecipitation of EV’s isolated from human plasma.

Article Snippet: Immunoblotting was performed using specific antibodies for CD9 and CD163 (pAb rabbit anti-human CD9; Systems Bioscience, mAb rat anti-mouse CD9 (clone MF1); Bio-Rad, UK, mAb mouse anti-human CD163 (clone Mac2–158); IQ products, Netherlands and pAb rabbit anti-mouse CD163) and a secondary HRP conjugated goat anti-rabbit, rat or mouse IgG antibody (Sigma-Aldrich).

Techniques: Staining, Isolation, Clinical Proteomics, Purification, Immunoprecipitation

Journal: Scientific Reports

Article Title: Soluble ectodomain CD163 and extracellular vesicle-associated CD163 are two differently regulated forms of ‘soluble CD163’ in plasma

doi: 10.1038/srep40286

Figure Lengend Snippet: Levels of sCD163 measured by CD163 ELISA in total plasma, EV depleted plasma (Exoquick treated) and membrane protein depleted plasma (aqueous phase from Tx-114 treatment) in human volunteers (n = 15, males, age 20–27, mean 22.8) after an i.v. bolus injection of endotoxin (4 ng/kg). Data presented as median with interquartile range prior to, 1 h and 24 h after intravenous endotoxin injection.

Article Snippet: Immunoblotting was performed using specific antibodies for CD9 and CD163 (pAb rabbit anti-human CD9; Systems Bioscience, mAb rat anti-mouse CD9 (clone MF1); Bio-Rad, UK, mAb mouse anti-human CD163 (clone Mac2–158); IQ products, Netherlands and pAb rabbit anti-mouse CD163) and a secondary HRP conjugated goat anti-rabbit, rat or mouse IgG antibody (Sigma-Aldrich).

Techniques: Enzyme-linked Immunosorbent Assay, Clinical Proteomics, Membrane, Injection

Journal: Scientific Reports

Article Title: Soluble ectodomain CD163 and extracellular vesicle-associated CD163 are two differently regulated forms of ‘soluble CD163’ in plasma

doi: 10.1038/srep40286

Figure Lengend Snippet: Total sCD163 (untreated plasma), soluble ectodomain CD163 (aqueous phase of Tx-114-treated plasma) and membrane-associated CD163 (detergent phase of Tx-114-treated plasma) in plasma of the healthy volunteers vs. patients with severe sepsis. Data presented as median with interquartile range and *p < 0.05 and ****p < 0.0001 in non-parametric Mann-Whitney t-test.

Article Snippet: Immunoblotting was performed using specific antibodies for CD9 and CD163 (pAb rabbit anti-human CD9; Systems Bioscience, mAb rat anti-mouse CD9 (clone MF1); Bio-Rad, UK, mAb mouse anti-human CD163 (clone Mac2–158); IQ products, Netherlands and pAb rabbit anti-mouse CD163) and a secondary HRP conjugated goat anti-rabbit, rat or mouse IgG antibody (Sigma-Aldrich).

Techniques: Clinical Proteomics, Membrane, MANN-WHITNEY

Journal: Scientific Reports

Article Title: Soluble ectodomain CD163 and extracellular vesicle-associated CD163 are two differently regulated forms of ‘soluble CD163’ in plasma

doi: 10.1038/srep40286

Figure Lengend Snippet: ( A ) Correlation of total sCD163 (left) soluble ectodomain CD163 (middle) and membrane-associated CD163 (right) level measured by CD163 ELISA in plasma and SOFA score. Correlation was tested by Spearman’s non-parametric correlation analysis. ( B ) membrane-associated CD163 levels in plasma of septic patients with SOFA score <10 (mortality risk <50%) and SOFA ≥ 10 (mortality risk >50%). Data presented as median with interquartile range and ***p < 0.001 as tested by the non-parametric Mann-Whitney t-test.

Article Snippet: Immunoblotting was performed using specific antibodies for CD9 and CD163 (pAb rabbit anti-human CD9; Systems Bioscience, mAb rat anti-mouse CD9 (clone MF1); Bio-Rad, UK, mAb mouse anti-human CD163 (clone Mac2–158); IQ products, Netherlands and pAb rabbit anti-mouse CD163) and a secondary HRP conjugated goat anti-rabbit, rat or mouse IgG antibody (Sigma-Aldrich).

Techniques: Membrane, Enzyme-linked Immunosorbent Assay, Clinical Proteomics, MANN-WHITNEY

Journal: Scientific Reports

Article Title: Soluble ectodomain CD163 and extracellular vesicle-associated CD163 are two differently regulated forms of ‘soluble CD163’ in plasma

doi: 10.1038/srep40286

Figure Lengend Snippet: ( A ) CD9 and CD163 Western blot analysis of mouse plasma before (total serum) and after (non-EV fraction) precipitation of EVs (EV-fraction). Blots are cropped for presentation. Full-length blots are available in . ( B ) Beads-based FACS analysis of exosome-FITC and CD9 or CD163 co-staining on αCD163/αCD9 (dark grey) or isotype IgG immuno-precipitated EVs isolated from mouse plasma. ( C ) Levels of ectodomain sCD163 and EV-CD163 measured by mouse CD163 ELISA in Tx-114 phase-separated plasma of mice (n = 6) in models of endotoxemia, sterile thioglycollate-induced peritonitis and Listeria Monocytogenes bacteraemia.

Article Snippet: Immunoblotting was performed using specific antibodies for CD9 and CD163 (pAb rabbit anti-human CD9; Systems Bioscience, mAb rat anti-mouse CD9 (clone MF1); Bio-Rad, UK, mAb mouse anti-human CD163 (clone Mac2–158); IQ products, Netherlands and pAb rabbit anti-mouse CD163) and a secondary HRP conjugated goat anti-rabbit, rat or mouse IgG antibody (Sigma-Aldrich).

Techniques: Western Blot, Clinical Proteomics, Staining, Isolation, Enzyme-linked Immunosorbent Assay, Sterility

Journal: European Journal of Histochemistry : EJH

Article Title: Extracellular vesicles from bone mesenchymal stem cells transport microRNA-206 into osteosarcoma cells and target NRSN2 to block the ERK1/2-Bcl-xL signaling pathway

doi: 10.4081/ejh.2022.3394

Figure Lengend Snippet: Identification of BMSCs and EVs. The BMSCs obtained from ATCC were subcultured. A) The morphology of BMSCs in the fourth generation was observed by microscope. B) The positive marker proteins (CD29, CD44, and CD71) were positively expressed and negative marker proteins (HLA-DR, CD34, and CD45) of BMSCs were negatively expressed detected using flow cytometry. C) Lipid differentiation was observed by oil red O staining. D) Alizarin red staining was used to observe osteogenic differentiation; the EVs were extracted. E) The morphology of BMSCs-EVs was observed with a typical cup shape by TEM. F) The size distribution of EVs was mainly at about 100 nm analyzed using NTA. G) WB detected that the expressions of EV surface CD9, CD63, and CD81 enrichment and calnexin protein were not significantly expressed.

Article Snippet: The BMSCs-EVs were identified by the following methods: i) The morphology of isolated EVs was observed using transmission electron microscopy (TEM); ii) The EV size distribution was analyzed using nanoparticle tracking analysis (NTA); iii) Western blot (WB) was used to verify the expressions of BMSC-EVs surface positive markers CD9 (ab236630, 1/1000; Abcam), CD63 (ab134045, 1/1000; Abcam), and CD81 (ab109201, 1/1000; Abcam), and a negative marker Calnexin (ab92573, 1/20000; Abcam), and the supernatant of BMSCs added with GW4869 was used as negative control (NC).

Techniques: Microscopy, Marker, Flow Cytometry, Staining