TSG101 Antibody - #DF8427

Fig. 3 CD45 shed from WBCs could be transferred via EVs to tumor cells. a GFP+ Caco2 cells co-cultured with Jurkat cells at a ratio of 1:4 for different time (0, 1, 2, 4, 8, 16 h), then GFP+ Caco2 cells were collected and stained with anti-CD45-APC to dynamically analyze the MFI shifting of CD45 among GFP+ Caco2 cells using flow cytometry. b Flow cytometric analysis of CD45 transfer from Jurkat cells to Caco2 cells, or from Jurkat cells/ THP1 cells/ WBCs to DLD1 cells following 16 h of indirect co-culture. c, d Bar graph showing the MFI of CD45-PE of Caco2 cells or DLD1 cells after 16 h of indirect co-culture with Jurkat cells/ THP1 cells/ WBCs. e, f Immunoblot analysis showing the makers of EVs isolated from supernatant of THP1 and Jurkat cells. e Density gradient fractions of EVs from Jurkat cells. f Positive (TSG101 and CD63) and negative (Calnexin) markers of EVs. WCL: whole cell lysis. Each ladder was loaded with protein 30 μg. g Nanoparticle tracking analysis of Jurkat cell-derived EVs. h Transmission electron microscope imaging of Jurkat cell-derived EVs. Scale bar = 50 nm. i Plasma EVs charactering by immunoblotting. Jurkat-EVs was used as positive control. P1- and P2-EVs were examples of two CRC cancer patients, HD1 and HD2 were examples of two healthy donors. j Plasma EVs charactering by nano-flow cytometry analysis. k, l CD45 ELISA assay for the measurement of EVs-derived CD45 from healthy donors (HD) and CRC patients (PD) with or without metastasis. m Time series of live cell imaging of DLD1-RFP cells after the addition of EVs (20 μg/mL) derived from CD45-GFP-expressing HEK293T cells for up to 8 h. Scale bar = 5 μm. n Immunofluorescence images of GFP+ Caco2 cells after incubation with PBS or EVs isolated from Jurkat cell supernatant for 12 h. Left scale bar = 20 μm, right scale bar = 5 μm. o Flow cytometric analysis for the measurement of Jurkat cell-derived EVs uptake by Caco2 cells. Caco2 cells were incubated with different concentration of PKH26-stained EVs (0, 1, 2, or 4 μg/mL) for 12 h before analysis. p Caco2 cells were incubated with PKH26-stained EVs (2 μg/mL) and chlorpromazine (CPZ, 0, 5, 10, or 20 μg/mL) for 12 h before collection for flow cytometric analysis of PKH26+ Caco2 cells. All bar graph data are presented as means ± SEM. *P 

Figure 1 Characterization of exosomes: the size distributions of blank exosome (A) and exosome-doxorubicin (B) measured by NTA. The mean particle diameters were 141.6 nm for free exosome and 178.1 nm for exosome-doxorubicin. The morphology of blank exosome (C) and exosome-doxorubicin (D) as observed by TEM. (E) Western blotting analysis of the exosomal proteins CD81 and TSG101.

FIGURE 1Cell identification and exosome characterization. (A) Representative flow cytometry histograms of BMSCs and ADSCs show positive staining for CD29 and CD90 but not for CD34 and CD45. (B) BMSCs and ADSCs were successfully induced into osteoblasts (positively stained with Alizarin Red S) and adipocytes (positively stained with Oil Red O). The magnification is 100×. (C) Representative immunofluorescence results of CCSMCs show positive expression for α-SMA and desmin. Scale bars = 50 μm. (D) Exosomes derived from CCSMCs, BMSCs and ADSCs were observed using transmission electron microscopy, and the particle size distributions of the exosomes were measured by nanoparticle tracking analysis. Scale bars = 100 nm. (E) Representative results of Western blot analysis of exosomes derived from CCSMCs, BMSCs and ADSCs show positive expression for CD9, CD63 and TSG101 but not for calnexin. CCSMC: corpus cavernosum smooth muscle cell; BMSC: bone marrow stem cell; ADSC: adipose-derived stem cell; CCSMC-EXOs: exosomes derived from corpus cavernosum smooth muscle cells; BMSC-EXOs: exosomes derived from bone marrow stem cells; ADSC-EXOs: exosomes derived from adipose-derived stem cells; α-SMA: α-smooth muscle actin; DAPI: 4’,6-diamidino-2-phenylindole

Figure 1 Isolation of TRAIL-expressing extracellular vesicles (EV-T) and post-loading of Dinaciclib (Dina) into EV-T to prepare the complexed therapeutics (EV-T-Dina). (a) Isolation of extracellular vesicles (EVs) by ultracentrifugation; (b) Examination of isolated EV-Ts by transmission electron microscopy (TEM); (c) EV-T analysis by high sensitivity flow cytometry; (d) Assessment of TRAIL expression in EV-Ts by a commercial human TRAIL-specific ELISA kit; (e) Expressional detection of TRAIL and the EV biomarker TSG101 in EVs and EV-Ts by immunoblotting. Uncropped blots are available in Figure S2; (f) Detection of Dina by high-performance liquid chromatography (HPLC); (g) Establishment of a linear regression curve and function for the detection of Dina; (h) Determination and comparison of Dina encapsulation rates by three loading methods of mixing, amphotericin B (AmB), and sonication. Values are means ± SD (n = 3), * p < 0.05, ** p < 0.01, by one-way ANOVA/Bonferroni multiple-comparison post hoc test.

Figure 1. Characterisation of EVs derived from the serum and plasma of NSCLC patients and controls. (a) The shape and structure of serum and plasma EVs isolated by EXOquick kit under TEM. The red arrow represents EVs with typical characteristics (scale bars are 200 nm). (b) The size of EVs derived from control groups and NSCLC groups was analysed by NTA. (c) Western blots of EVs membrane markers, including Alix, CD63, TSG101, CD9, and one negative marker ALB.