Journal: Journal of Extracellular Vesicles

Article Title: Human Endometriotic Lesion‐Derived Small Extracellular Vesicles Impair Macrophage Function in the Peritoneal Microenvironment

doi: 10.1002/jev2.70227

Figure Lengend Snippet: Characterisation of EEO‐sEVs. (a) The mode size, mean size and concentration of EEO‐sEVs derived from the EUT of CON and EM patients, and the ECTs of EM were analysed by NTA. Data are shown as mean ± SD. (b) Flow cytometry analysis of CD81, CD9 and CD63 expression in EEO‐sEVs derived from EUT (CON), EUT (EM) and ECT (EM) ( n = 3). Marker expression was quantified by MFI. (c) Western blot analysis of syntenin and AGO‐1 (argonaute‐1) expression in pooled EUT (CON + EM) and ECT (EM) EEO‐sEVs ( n = 3) compared to EEO cell lysates. (d) Representative TEM image of EEO‐sEVs derived from EUT (CON) and EUT (EM) and ECT (EM). Scale bar, 200 nm. (e, f) NTA of mode size, mean size and concentration of EVs populations isolated from EEO (EUT EM and ECT EM) collected from 72 h incubation (e) at Days 4–7 and 7–10 at P3 ( n = 3) and (f) at P3 and P4 ( n = 4). Data are shown as mean ± SD. (g, h) Surface marker profiles of EEO‐sEVs (EUT EM and ECT EM) collected from 72 h incubation at (g) Days 4–7 and 7–10 at Passage 3 (P3) ( n = 3), and (h) at P4 was analysed by flow cytometry. The MFI (MACSPlex) was normalised by average MFI of CD81, CD9 and CD63. Data are shown as mean nMFI ± SD. ARG‐1, argonaute‐1; CON, control; ECT, ectopic epithelium; EEO, endometrial epithelial organoid; EM, endometriosis; EUT, eutopic epithelium; MFI, median fluorescence intensity; sEV, small extracellular vesicle.

Article Snippet: sEV surface proteins were characterised using the MACSPlex EV kit IO (Miltenyi Biotec, Germany) following the manufacturer's instructions.

Techniques: Concentration Assay, Derivative Assay, Flow Cytometry, Expressing, Marker, Western Blot, Isolation, Incubation, Control, Fluorescence

Journal: Journal of Extracellular Vesicles

Article Title: Human Endometriotic Lesion‐Derived Small Extracellular Vesicles Impair Macrophage Function in the Peritoneal Microenvironment

doi: 10.1002/jev2.70227

Figure Lengend Snippet: ECT EEO has distinct EV surface profiles (MACSPlex analysis). (a) Heatmap indicating hierarchical clustering analysis of nMFI surface marker expression on EEO‐sEV among EUT (CON), EUT (EM) and ECT (EM). The MFI was normalised by average MFI of CD81, CD9 and CD63. Patient IDs are indicated at the bottom; * denotes paired ECT EEO‐sEVs samples from the same EM patient. (b) PCA of surface marker profiles in EEO‐sEVs. PC1 versus PC2 scores plot showing sample distribution across CON (green), EUT (blue) and ECT (red) groups. Ellipses represent 95% confidence intervals. Paired EUT and ECT samples are indicated by matching numbers with * on ECT, connected by black dashed arrows. (c) Top 15 surface markers contributing to PC2 variance in the PCA. Positive loadings (red) indicate markers with higher expression in the positive direction of PC2, while negative loadings (blue) indicate markers with higher expression in the negative direction. The length of each bar represents the magnitude of contribution. (d–g) The nMFI of (d) CD29, (e) CD44, (f) CD41b and (g) EpCAM expression on EEO‐sEVs from EUT (CON), EUT (EM) and ECT (EM). Data are presented as mean ± SD. * p < 0.05, ** p < 0.01. CON, control; ECT, ectopic epithelium; EEO, endometrial epithelial organoid; EM, endometriosis; EUT, eutopic epithelium; MFI, median fluorescence intensity; sEV, small extracellular vesicle.

Article Snippet: sEV surface proteins were characterised using the MACSPlex EV kit IO (Miltenyi Biotec, Germany) following the manufacturer's instructions.

Techniques: Marker, Expressing, Control, Fluorescence

Journal: Journal of Extracellular Vesicles

Article Title: Human Endometriotic Lesion‐Derived Small Extracellular Vesicles Impair Macrophage Function in the Peritoneal Microenvironment

doi: 10.1002/jev2.70227

Figure Lengend Snippet: PF‐sEVs are derived from various cell origins. (a) MFI of CD81, CD9 and CD63 expression on sEVs isolated from the PF of individual CON and EM patients. CTRL represents the negative control of MACSPLEX capture beads incubated with PBS with no PF‐sEVs. (b) Western blot analysis of syntenin and albumin expression in EEO‐sEVs isolated from CON and EM ( n = 3) compared to PF cell lysates. (c) Representative TEM image of PF‐sEVs derived from CON and EM. Scale bar, 1000 nm. (d, e) NTA of PF‐sEVs shows the mode size, mean size and concentration of PF‐sEVs derived from CON, EMI/II and EMIII/IV stage patients (d) and proliferative versus secretory phase of the menstrual cycle (e). CON, control; EM, endometriosis; MFI, median fluorescence intensity; PF, peritoneal fluid; pMΦ, peritoneal macrophage; sEV, small extracellular vesicle.

Article Snippet: sEV surface proteins were characterised using the MACSPlex EV kit IO (Miltenyi Biotec, Germany) following the manufacturer's instructions.

Techniques: Derivative Assay, Expressing, Isolation, Negative Control, Incubation, Western Blot, Concentration Assay, Control, Fluorescence

Journal: Journal of Extracellular Vesicles

Article Title: Human Endometriotic Lesion‐Derived Small Extracellular Vesicles Impair Macrophage Function in the Peritoneal Microenvironment

doi: 10.1002/jev2.70227

Figure Lengend Snippet: PF‐sEV surface marker characterisation (MACSPlex analysis). (a) Heatmap indicating hierarchical clustering analysis of nMFI surface marker expression on PF‐sEVs. (b) PCA of surface marker profiles in PF‐sEVs. PC1 versus PC2 scores plot showing sample distribution across CON (blue), EM I/II (green) and EM III/IV (red) groups. Ellipses represent 95% confidence intervals. (c) Violin plots showing the distribution of PC2 scores across groups. Black bars indicate median values and quartiles. (d) Top 10 surface markers contributing to PC2 variance in the PCA. Positive loadings (red) indicate markers with higher expression in the positive direction of PC2, while negative loadings (blue) indicate markers with higher expression in the negative direction. The length of each bar represents the magnitude of contribution. (e) Venn diagram showing the distribution and overlap of surface markers on sEVs derived from pMΦ and EEO. CON, control; EEO, endometrial epithelial organoid; EM, endometriosis; nMFI, normalised median fluorescence intensity; PCA, principal component analysis; PF, peritoneal fluid; pMΦ, peritoneal macrophage; sEV, small extracellular vesicle.

Article Snippet: sEV surface proteins were characterised using the MACSPlex EV kit IO (Miltenyi Biotec, Germany) following the manufacturer's instructions.

Techniques: Marker, Expressing, Derivative Assay, Control, Fluorescence

Journal: Journal of Extracellular Vesicles

Article Title: Human Endometriotic Lesion‐Derived Small Extracellular Vesicles Impair Macrophage Function in the Peritoneal Microenvironment

doi: 10.1002/jev2.70227

Figure Lengend Snippet: sEV mediates phagocytic suppression via CD47 in endometriosis. (a) PF‐sEVs from control ( n = 5) and women with endometriosis ( n = 5) were analysed for CD47 coexpression using the MACSPlex EV Kit IO. The data were normalised by fold change, calculated as the ratio of MFI from EV‐positive beads to that of PBS‐only negative control beads for each marker. (b) The Venn diagram of coexpressed markers with CD47 on PF‐sEV between CON ( n = 5) and EM ( n = 5). (c) Flow cytometry analysis of the effect of blocking CD47 on PF‐sEV on regulating macrophage phagocytosis. THP‐1‐derived macrophages were treated with PF‐sEV from EM or CON, preincubated with either IgG (control) or anti‐CD47 antibodies. Phagocytic activity was assessed using pHrodo E. coli deep red bioparticles. Fold change was calculated by dividing the deep red MFI of PF‐sEV‐treated cells by the mean deep red MFI of untreated cells from three wells. Data are shown as mean ± SD. * p < 0.05, ** p < 0.01. CON, control; ECT, ectopic epithelium; EEO, endometrial epithelial organoid; EM, endometriosis; EUT, eutopic epithelium; MFI, median fluorescence intensity; PF, peritoneal fluid; sEV, small extracellular vesicle.

Article Snippet: sEV surface proteins were characterised using the MACSPlex EV kit IO (Miltenyi Biotec, Germany) following the manufacturer's instructions.

Techniques: Control, Negative Control, Marker, Flow Cytometry, Blocking Assay, Derivative Assay, Activity Assay, Fluorescence