CD9 Antibody - #AF5139

Figure 2 Characterization of ESIONPs@EXO derived from ESIONPs engineered macrophages. (A) The morphology of EXO and ESIONPs@EXO determined by TEM. Scale bar: 200 nm (left) and 100 nm (right). (B) The size distribution of EXO and ESIONPs@EXO evaluated by NTA. (C) Western blot analysis of CD9, CD63, CD81, TSG101, and calnexin. (D) Relaxation properties of ESIONPs@EXO. (E) T1 and T2 weighted MR images of ESIONPs@EXO at different concentrations (measured on a 3 T MR scanner). 1/T1 (F) and 1/T2 (G) relaxation rates of ESIONPs@EXO at different concentrations.

Figure 2 Preparation and characterization of ExoIRES-IL-10. (A) Schematic of the exosomes preparation and isolation process. (B) Western blot analysis of the exosomal inclusive markers (TSG101, CD9), exclusive marker (GM130), negative markers to confirm the purity (APOA1) in the isolated exosomes and parental cells. HEK293T cells were treated with PBS or transfected, empty vector, or IRES-Il-10 plasmids. (C) Representative transmission electron microscope (TEM) images of ExoNone, ExoEmpty or ExoIRES-IL-10. Scale bar = 200 nm. (D) Size distribution of the isolated exosomes as indicated. (E) qPCR analysis of Il-10 mRNA in the isolated exosomes as indicated. GAPDH served as an internal control. (F) qPCR analysis of Il-10 mRNA in ExoIRES-IL-10 in exosomal RNA degradation assay as indicated. GAPDH served as an internal control. Data are presented as mean ± SEM of three independent experiments. ND, not determined as Ct value greater than 38.

Fig. 1 Construction and features of engineered CAP-Nrf2-Exos. (A, B) Western blot and qRT‒PCR analysis showing the mRNA and protein expression levels of Nrf2 in HEK293T cells. (C, D) Western blot and qRT‒PCR analysis of Nrf2 in the engineered exosomes. (E, F) Western blot and qRT‒PCR analysis of Nrf2 expression in CEP cells after treatment with engineered exosomes. (G) Western blot analysis of Lamp2b, CD9, CD63, TSG101, and β-Actin in HEK293T cells, HEK293T cells transfected with CAP-Nrf2 overexpression plasmids, Exos and CAP-Nrf2-Exos. (H) Exos and CAP-Nrf2-Exos were examined by NTA. (I) TEM analysis of Exos and CAP-Nrf2-Exos isolated from the culture medium of HEK293T cells. Scale bar, 100 nm. (J) Fluorescence images of PKH26-labelled Exos and CAP-Exos internalized by CEP cells. Scale bar, 50 μm and 10 μm. (K) Fluorescence images of DiR-labelled Exos and CAP-Exos in the CEP and IVD in the groups mentioned above. (n = 3, *p 

Fig. 1 Identification and characterization of DPSCs-sEV. a The morphology of DPSCs-sEV was determined by TEM, scale bar = 50 nm. b Expression of CD9 and CD63 in the DPSCs-sEV (sEV represents DPSCs-sEV and Lys represents DPSCs lysate, 尾-actin is a control for the lysate). c Nano-flow cytometry showed DPSCs-sEV ranged between 30 and 150 nm in diameter. d PKH26-labeled DPSCs-sEV were found in macrophage cytosol

Figure 2. Identification of EVs derived from LPS-stimulated DPCs (LPS-EVs) and establishment of in vitro model. (a) CCK8 assay detected cell viability of DPCs treated LPS (0.1, 1, 10, 100 µg/mL). (b) 1 µg/mL LPS increased EVs secretion of DPCs, compared to the other groups. (c) Schematic diagram shows the four types EV collected from EV-free culture medium of DPCs with or without LPS stimulation (0.1, 1, 10 µg/mL), which were used in subsequent assays. (d) Nanoparticle tracing assay (NTA) revealed the diameter of collected EVs was approximately 100 nm. (e) Scanning electron microscopy (SEM) of cup and saucer-shaped EVs. (f) Immunofluorescence staining confirmed that PKH26-labeled EVs (red) were endocytosed by SCAPs. DAPI (blue), and F-actin (green). (g) Western blot analysis of EVs surface markers (CD9, CD63, CD81 and TSG101). (h) CCK8 assay detected the effect of Nor-EV and LPS-EVs on cell viability of SCAPs. *p < 0.05. **p < 0.01. ***p < 0.001.#p < 0.0001.

Figure 1.Isolation of EVs and determination of RIF-EVs and FER-EVs. (A) Western blotting shows that RIF-EVs and FER-EVs expressed classic EV protein markers Alix, TSG101, and CD9. Representative shapes of (B) RIF-EVs and (C) FER-EVs detected with the use of transmission electron microscopy. The size and distribution of (D) RIF-EVs and (E) FER-EVs examined with the use of nanoparticle tracking analysis. ECs ¼ endometrial cells; EVs ¼ extracellular vesicles; FER ¼ fertile women; RIF ¼ women with recurrent implantation failure. Liu. Extracellular vesicles regulate embryos. Fertil Steril 2020.