shigella flexneriae  (ATCC)

Journal: Journal of Extracellular Vesicles

Article Title: Single extracellular vesicle analysis in human amniotic fluid shows evidence of phenotype alterations in preeclampsia

doi: 10.1002/jev2.12217

Figure Lengend Snippet: Characterization of NP‐EVs. (a) Nanoparticle tracking analysis of NP‐EVs ( n = 30) showing homogenous size and concentration. Dilution of 1:100 in PBS in 1ml was used to test the samples (left panel). A representative graph showing size distribution of a NP‐EV sample (right panel). (b) Representative Western blot image showing the presence of CD63, CD81, CD105 and HLA‐G in NP‐EVs (10–15 μg proteins) and in AFSC lysate (10–15 μg proteins) and the presence of calreticulin in cell lysate only. Three different NP‐EV samples were tested with similar results. (c) Representative transmission electron microscopy images at low‐ and high‐power fields of NP‐EVs showing heterogenous EV population. (d) Representative super‐resolution microscopy images of single amniotic fluid‐derived EVs expressing CD63 (red) and HLA‐G (green). The number of single and double positive EVs for CD63 and HLA‐G was analysed in three NP‐EV preparations using the CODI software; the graph shows the mean ± SD of a cumulative analysis of 10 fields for each preparation, total EV number: 6676

Article Snippet: Primary antibodies used were: CD81 at 1:200 (SC‐31234), CD63 at 1:200 (SC‐5275), HLA‐G at 1:200 (SC‐21799), (all from Santa Cruz), CD105 at 1:1000 (362‐820, Ancell) and at 1:2000 calreticulin (2891S, Cell Signaling Technology, Danvers, MA, USA).

Techniques: Concentration Assay, Western Blot, Transmission Assay, Electron Microscopy, Super-Resolution Microscopy, Derivative Assay, Expressing, Software

Journal: Journal of Extracellular Vesicles

Article Title: Single extracellular vesicle analysis in human amniotic fluid shows evidence of phenotype alterations in preeclampsia

doi: 10.1002/jev2.12217

Figure Lengend Snippet: Increased CD105 expression in PE‐EVs. (a) MACSPlex analysis showing the median fluorescence intensity of surface markers characteristic of EVs (tetraspanins) or different cell of origin, expressed by NP‐ and PE‐EVs ( n = 3). Expression of CD105 and SSEA‐4 was significantly increased in PE‐EVs. 5.8 × 10 8 EVs were analysed, diluted to a final volume of 120 μl of MACSPlex buffer. (b) Diagram explaining the experimental method behind ExoView technology in relation to the graphs in panel c. (c and d) ExoView analysis of amniotic fluid‐derived NP‐EVs ( n = 6) and PE‐EVs ( n = 6). (c) Comparison of the expression of HLA‐G, Tie‐2, CD105 and CD117 (c‐kit) shown as average fluorescent particle count in NP‐EVs vs PE‐EVs from combined tetraspanins capture of CD63, CD81 and CD9. (d) Normalized expression of HLA‐G positive EVs co‐expressing other angiogenic (CD105 and Tie‐2) and stem cell (CD117) markers. 5.8 × 10 8 EVs in final volume of 35 μl of buffer were used for all samples. Unpaired student's t ‐test:* = P < 0.05, * * = P < 0.01, *** = P < 0.001

Article Snippet: Primary antibodies used were: CD81 at 1:200 (SC‐31234), CD63 at 1:200 (SC‐5275), HLA‐G at 1:200 (SC‐21799), (all from Santa Cruz), CD105 at 1:1000 (362‐820, Ancell) and at 1:2000 calreticulin (2891S, Cell Signaling Technology, Danvers, MA, USA).

Techniques: Expressing, Fluorescence, Derivative Assay, Comparison

Journal: Journal of Extracellular Vesicles

Article Title: Single extracellular vesicle analysis in human amniotic fluid shows evidence of phenotype alterations in preeclampsia

doi: 10.1002/jev2.12217

Figure Lengend Snippet: Characterization of HLA‐G‐expressing EVs from term AFSCs. (a) Diagram explaining the experimental method in relation to the graph in panel b. (b) Fluorescent particle count of AFSC‐EVs captured on tetraspanin‐coated chip analysed by ExoView, showing expression of HLA‐G + and CD105 + EVs, but lack of co‐expression of the markers. (c) Graphical representation of MASCPlex analyses to characterize HLA‐G + AFSC‐EVs, using Ab‐coated fluorescent beads and APC‐labelled anti‐HLA‐G Ab. (d) The graph shows the median fluorescence intensity of surface markers co‐expressed on HLA‐G + AFSC‐EVs ( n = 3). AFSC‐EVs (5.8 × 10 8 ) were gated for the HLA‐G positivity and analysed for the expression of a panel of markers. No expression of CD105 was detected. Illustrations in panels A and C were created with https://biorender.com

Article Snippet: Primary antibodies used were: CD81 at 1:200 (SC‐31234), CD63 at 1:200 (SC‐5275), HLA‐G at 1:200 (SC‐21799), (all from Santa Cruz), CD105 at 1:1000 (362‐820, Ancell) and at 1:2000 calreticulin (2891S, Cell Signaling Technology, Danvers, MA, USA).

Techniques: Expressing, Fluorescence

Journal: Journal of Extracellular Vesicles

Article Title: Single extracellular vesicle analysis in human amniotic fluid shows evidence of phenotype alterations in preeclampsia

doi: 10.1002/jev2.12217

Figure Lengend Snippet: Effect of NP‐EVs and PE‐EVs on tube formation and role of CD105. (a) Quantification of tube lengths of HUVEC treated with NP‐EVs, PE‐EVs with or without anti‐CD105 Ab (TRC‐105) (8 μg/ml). A concentration of 1000 EVs/cell was used. Positive control: complete EBM medium; negative control: EBM medium without FBS. s‐Endoglin (100 ng/ml) was used as control for angiogenesis inhibition. Statistical analysis was performed using ANOVA with Bonferroni's post‐hoc test. * = P < 0.05, ** = P < 0.01, *** = P < 0.001. (b) Representative images of the tube formation assay

Article Snippet: Primary antibodies used were: CD81 at 1:200 (SC‐31234), CD63 at 1:200 (SC‐5275), HLA‐G at 1:200 (SC‐21799), (all from Santa Cruz), CD105 at 1:1000 (362‐820, Ancell) and at 1:2000 calreticulin (2891S, Cell Signaling Technology, Danvers, MA, USA).

Techniques: Concentration Assay, Positive Control, Negative Control, Control, Inhibition, Tube Formation Assay

Journal: Journal of Extracellular Vesicles

Article Title: Single extracellular vesicle analysis in human amniotic fluid shows evidence of phenotype alterations in preeclampsia

doi: 10.1002/jev2.12217

Figure Lengend Snippet: Graphical illustration summarizing the main findings. The figure shows the multiple possible sources of term amniotic fluid‐derived EVs and the main changes in PE‐EVs. Their antiangiogenic properties are supported by the specific upregulation of CD105 surface. The image was created with https://biorender.com

Article Snippet: Primary antibodies used were: CD81 at 1:200 (SC‐31234), CD63 at 1:200 (SC‐5275), HLA‐G at 1:200 (SC‐21799), (all from Santa Cruz), CD105 at 1:1000 (362‐820, Ancell) and at 1:2000 calreticulin (2891S, Cell Signaling Technology, Danvers, MA, USA).

Techniques: Derivative Assay