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.