Journal: Scientific Reports

Article Title: Active macropinocytosis induction by stimulation of epidermal growth factor receptor and oncogenic Ras expression potentiates cellular uptake efficacy of exosomes

doi: 10.1038/srep10300

Figure Lengend Snippet: ( a ) Confocal microscopic observation of A431 cells treated with CD63-GFP-exosomes (20 μg/ml) in the presence or absence of EGF (500 nM) for 24 h at 37 °C. Green signals, CD63-GFP-exosomes; blue signals, Hoechst 33342 for nuclear staining. Scale bar, 20 μm. ( b ) Relative cellular uptake of CD63-GFP-exosomes in the same experimental condition of ( a ) analysed using a flow cytometer. ( c ) Internalisation of CD63-exosomes (20 μg/ml) and Texas Red-dextran (70 kDa, macropinocytosis marker) in the presence of EGF (500 nM) by A431 cells analysed using a confocal microscopy after treatment for 24 h at 37 °C. Arrows show representative colocalisation of exosomes and dextran inside cells. Scale bar, 10 μm. ( d ) Effects of the macropinocytosis inhibitor, EIPA (100 nM), on the cellular uptake of CD63-GFP-exosomes (20 μg/ml) with EGF (100 nM) for 3 h at 37 °C, analysed using a flow cytometer. ( b,d ) The data are the averages (±SD) of three experiments. ** p < 0.01, *** p < 0.001.

Article Snippet: The boiled samples were separated by 10% SDS-PAGE, transferred to polyvinylidene fluoride (PVDF) membranes (GE Healthcare, Pittsburgh, PA, USA), and treated with anti-CD63 antibody (TS63, Abcam, Cambridge, UK).

Techniques: Staining, Flow Cytometry, Marker, Confocal Microscopy

Journal: Scientific Reports

Article Title: Active macropinocytosis induction by stimulation of epidermal growth factor receptor and oncogenic Ras expression potentiates cellular uptake efficacy of exosomes

doi: 10.1038/srep10300

Figure Lengend Snippet: ( a ) Relative cellular uptake of CD63-GFP-exosomes (20 μg/ml) in MIA PaCa-2 or BxPC-3 cells in the presence or absence of EGF (500 nM) for 24 h at 37 °C, analysed using a flow cytometer. ( b , c ) Relative cellular uptake of FITC-dextran ( b ) or FITC-transferrin ( c ) in MIA PaCa-2 or BxPC-3 cells in the absence of EGF for 24 h at 37 °C, analysed using a flow cytometer. ( a - c ) The data are the averages (±SD) of three experiments. * p < 0.05, ** p < 0.01, *** p < 0.001. ( d ) Confocal microscopic observation of MIA PaCa-2 cells treated with CD63-GFP-exosomes (20 μg/ml) in the presence of EGF (500 nM) in same experimental condition of ( a ). Green signals, CD63-GFP-exosomes; red signals, Texas Red-labelled dextran; blue signals, Hoechst 33342 for nuclear staining. Scale bar, 10 μm.

Article Snippet: The boiled samples were separated by 10% SDS-PAGE, transferred to polyvinylidene fluoride (PVDF) membranes (GE Healthcare, Pittsburgh, PA, USA), and treated with anti-CD63 antibody (TS63, Abcam, Cambridge, UK).

Techniques: Flow Cytometry, Staining

Journal: Scientific Reports

Article Title: Active macropinocytosis induction by stimulation of epidermal growth factor receptor and oncogenic Ras expression potentiates cellular uptake efficacy of exosomes

doi: 10.1038/srep10300

Figure Lengend Snippet: ( a ) Cell viability of A431 cells treated with CD63-GFP-exosomes (20 μg/ml) in serum-free cell culture medium with or without co-treatment of EGF (100 or 500 nM) for 24 h at 37 °C, analysed using a WST-1 assay. ( b ) Cytotoxicity of saporin-encapsulated exosomes (4 μg/ml) or saporin (7 μg/ml) with or without co-treatment of EGF (500 nM). A431 cells were treated with each test sample in 10% FBS containing cell culture medium for 48 h at 37 °C, prior to a WST-1 assay. The data are the averages (±SD) of three experiments. ** p < 0.01, *** p < 0.001.

Article Snippet: The boiled samples were separated by 10% SDS-PAGE, transferred to polyvinylidene fluoride (PVDF) membranes (GE Healthcare, Pittsburgh, PA, USA), and treated with anti-CD63 antibody (TS63, Abcam, Cambridge, UK).

Techniques: Cell Culture, WST-1 Assay

Journal: Scientific Reports

Article Title: Active macropinocytosis induction by stimulation of epidermal growth factor receptor and oncogenic Ras expression potentiates cellular uptake efficacy of exosomes

doi: 10.1038/srep10300

Figure Lengend Snippet: ( a ) Confocal microscopic observation of A431 cells treated with EGF- or transferrin-encapsulated CD63-GFP-exosomes (20 μg/ml) for 24 h at 37 °C. Green signals, CD63-GFP-exosomes; blue signals, Hoechst 33342 for nuclear staining. Scale bar, 20 μm. ( b ) Relative cellular uptake of CD63-GFP-exosomes with encapsulation of EGF or transferrin in the exosomes in the same experimental condition with ( a ), prior to analysis using a flow cytometer. The data represent the average (±SD) of three experiments. *** p < 0.001.

Article Snippet: The boiled samples were separated by 10% SDS-PAGE, transferred to polyvinylidene fluoride (PVDF) membranes (GE Healthcare, Pittsburgh, PA, USA), and treated with anti-CD63 antibody (TS63, Abcam, Cambridge, UK).

Techniques: Staining, Encapsulation, Flow Cytometry

Journal: Vaccine

Article Title: Gastric eosinophils are detrimental for Helicobacter pylori vaccine efficacy.

doi: 10.1016/j.vaccine.2021.05.034

Figure Lengend Snippet: Fig. 1. Comparative timeline analysis of eosinophils infiltration upon Hp infection of naïve mice. Eight week old Balb/c mice were infected with Hp. Eosinophils and/or Th cells were analyzed in the stomach, blood and spleen. (A) Gating strategies used in flow cytometry to characterize the absolute cell number and degranulation/activation status of eosinophils isolated from the stomach mucosa. (B) Analysis of stomach eosinophils and CD4+ Th cells. Total number of eosinophils (B.1), number of eosinophils expressing the degranulation marker CD63 (B.2), number of eosinophils expressing the activation marker PD-L1 and (B.3) total number of CD45+CD4+ Th cells (B.4). (C) Analysis of blood eosinophils. Total number of eosinophils (C.1), number of eosinophils expressing the degranulation marker CD63 (C.2), number of eosinophils expressing the activation marker PD-L1 (C.3). (D) Analysis of spleen eosinophils. Percentages of eosinophils (D.1), percentages of eosinophils expressing the degranulation marker CD63 (D.2), percentages of eosinophils expressing the activation marker PD-L1+ (D.3). ns, not significant; * p < 0.05; ** p < 0.01; *** p < 0.001 (Mann-Whitney test). Bar graphs show the mean ± SEM. For each time point, 7–8 mice were analyzed.

Article Snippet: Cells were then stained with anti-mouse antibodies to CD63- BrightTM fluorescein isothiocyanate (BrightTM FITC; Clone REA563, Miltenyi Biotec, Auburn, CA), CD11b-PerCP/Cyanine5.5 (PerCP/Cy5.5, Clone M1/70, BioLegend, San Diego, CA), PD-L1- Allophycocyanine (APC, Clone 10F.9G2, BioLegend), Ly6G-AlexaFluor 700 (AF700, Clone 1A8, BioLegend), CD4-APC/Cy7 (Clone GK1.5, BioLegend), CD45-VioBlue (Clone REA 737, Miltenyi biotec), Siglec-F- phycoerythrin (PE, Clone REA 798, Miltenyi biotec), CD11b-PacificBlueTM (Clone M1/70, BioLegend), MHC-II-AF700 (Clone M5/114.15.2, BioLegend), CD40-PE/Vio 615 (Clone REA965, Miltenyi biotec), CD3PerCP/Cy5.5 (Clone 17A2, BioLegend), CD45-AF700 (Clone 30-F11, BioLegend) for 20 min on ice.

Techniques: Infection, Cytometry, Activation Assay, Isolation, Expressing, Marker, MANN-WHITNEY

Journal: International Journal of Molecular Sciences

Article Title: Specific IgE and Basophil Activation Test by Microarray: A Promising Tool for Diagnosis of Platinum Compound Hypersensitivity Reactions

doi: 10.3390/ijms25073890

Figure Lengend Snippet: Allergological work-up outcome.

Article Snippet: After that, we added the biotinylated anti-rat CD63 antibodies (Clon REA444, Miltenyi Biotech, Bergisch Gladbach, Germany), diluted to 1:50 with WB for 30 min, and washed 3 times with WB.

Techniques:

Journal: International Journal of Molecular Sciences

Article Title: Specific IgE and Basophil Activation Test by Microarray: A Promising Tool for Diagnosis of Platinum Compound Hypersensitivity Reactions

doi: 10.3390/ijms25073890

Figure Lengend Snippet: Example of a positive BAT-FC in patient 6. Gating strategy for basophils: ( A ) doublet exclusion, FSC-H vs. FSC-A; basophils were identified as low SSC-A and CD123 + ( B ) and CD123 + HLA-DR neg cells ( C ). ( D ) The univariate histogram shows a positive BAT determined by an increased percentage of CD63 expression on the patient’s basophils with the highest concentration of oxaliplatin (250 µg/mL). ( E ) The univariate histogram (green) shows a positive BAT determined by increased mean fluorescence intensity (MFI) on the patient’s basophils at 250 µg/mL oxaliplatin; the gray histogram corresponds to the unstimulated cells. ( F ) Table showing the percentage of CD63+, MFI, and SI of CD203c in patient 6. Data were analyzed using FACSDiva software (verion 8.0.2).

Article Snippet: After that, we added the biotinylated anti-rat CD63 antibodies (Clon REA444, Miltenyi Biotech, Bergisch Gladbach, Germany), diluted to 1:50 with WB for 30 min, and washed 3 times with WB.

Techniques: Expressing, Concentration Assay, Fluorescence, Software

Journal: International Journal of Molecular Sciences

Article Title: Specific IgE and Basophil Activation Test by Microarray: A Promising Tool for Diagnosis of Platinum Compound Hypersensitivity Reactions

doi: 10.3390/ijms25073890

Figure Lengend Snippet: Examples of positive sIgE-microarray, BAT-microarray, and immunofluorescence detection of CD63 upon drug stimulation in patient 6, allergic to oxaliplatin, and patient 21, allergic to carboplatin. Cells were visualized under at ×100 magnification using a Olympus IX70 fluorescent microscope.

Article Snippet: After that, we added the biotinylated anti-rat CD63 antibodies (Clon REA444, Miltenyi Biotech, Bergisch Gladbach, Germany), diluted to 1:50 with WB for 30 min, and washed 3 times with WB.

Techniques: Microarray, Immunofluorescence, Microscopy

Journal: International Journal of Molecular Sciences

Article Title: Specific IgE and Basophil Activation Test by Microarray: A Promising Tool for Diagnosis of Platinum Compound Hypersensitivity Reactions

doi: 10.3390/ijms25073890

Figure Lengend Snippet: BAT-microarray immunoassay. The y -axis shows the average CD63 expression represented as average z -scores. The x -axis shows the drug concentration: oxaliplatin ( A ) and carboplatin ( B ) 1:100 molar ratio. The standardized fluorescence intensity represented as the average z -score was considered positive if it exceeded three (dotted line).

Article Snippet: After that, we added the biotinylated anti-rat CD63 antibodies (Clon REA444, Miltenyi Biotech, Bergisch Gladbach, Germany), diluted to 1:50 with WB for 30 min, and washed 3 times with WB.

Techniques: Microarray, Expressing, Concentration Assay, Fluorescence

Journal: Physiological Research

Article Title: Analysis of Gene Expression Profiles Regulating Phenotypic Transformation of Vascular Smooth Muscle Cells by Endothelial Cell-Derived Exosomes

doi: 10.33549/physiolres.935541

Figure Lengend Snippet: A ) TEM (×30,000, voltage = 100.0 kV) reveals that ECs exosomes have a double-concave disc structure. B ) NTA showed an average diameter of 85.45 nm, with 99.33 % of the exosomes at this size, consistent with typical extracellular vesicles (30–150 nm). C )WB analysis confirmed high expression of exosome marker proteins CD9, CD63, and CD81 in ECs exosomes.

Article Snippet: The complete medium for rat aortic ECs ( Procell Life Science & Technology Co., Ltd” (Wuhan, China, CM-R075)), it contained 5 % FBS, 50ng/mL VEGF, 10ng/mL EGF, 20ng/mL bFGF and 1 % penicillin and streptomycin,CD9, CD31, CD63, and CD81 monoclonal antibodies (Abcam, USA), fluorescein (Cy3)-labeled goat anti-rabbit IgG (Boster, Wuhan, China), exosome extraction kit (exoEasy Maxi Kit, Qiagen), GW4869 exosome inhibitor (Yensen, China), SW-CJ-IF clean bench, constant temperature CO 2 incubator (Thermo, USA), 6-well Transwell culture plate (0.4 mm) (Costar 3450, USA), Olympus inverted microscope, fluorescence microscope, Agilent 2100 Bioanalyzer (Agilent Technologies, USA), and NovaSeq 6000 (Illumina, USA) were employed in this investigation.

Techniques: Expressing, Marker