primary abs against cd63 and cd81 antibody  (Thermo Fisher)

Journal: International Journal of Molecular Sciences

Article Title: Umbilical Cord Mesenchymal Stem Cell-Derived Extracellular Vesicles Enhance Chondrocyte Function by Reducing Oxidative Stress in Chondrocytes

doi: 10.3390/ijms26167683

Figure Lengend Snippet: Characterization of UCMSCs and UCMSC-EVs. ( A ) Phase-contrast image of UCMSCs cultured on the plastic culture plate. Scale bar: 100 μm. ( B ) Representative histograms showing the expression of CD105, CD90, CD73, CD45, CD34, and HLA-DR on the surface of UCMSCs. Unstained cells were included as negative controls to validate the specificity of antibody staining. ( C ) Flow cytometry analysis to quantify the percentage of UCMSC-EVs positive for CD9, CD63, and CD81. ( D ) Western blot analysis of the protein expressions of CD63, CD81, and α-tubulin in UCMSCs and UCMSC-EVs. ( E ) Morphology of UCMSC-EVs (arrowhead), as observed using TEM.

Article Snippet: Overnight incubation at 4 °C was conducted using primary antibodies against CD81 (Proteintech, Rosemont, IL, USA, 66866-1-lg; 1:1000), CD63, and α-tubulin.

Techniques: Cell Culture, Expressing, Staining, Flow Cytometry, Western Blot

Journal: Journal of Extracellular Biology

Article Title: Evaluating the Influence of Different Serum‐Free Culture Conditions on the Production and Function of Natural Killer Cell‐Derived Extracellular Vesicles

doi: 10.1002/jex2.70049

Figure Lengend Snippet: EV phenotyping and functional analysis. (A) Equal amounts of EVs were captured on aldehyde/sulphate latex beads and stained with either isotype control antibody, anti‐CD63 or anti‐CD81 antibodies, and expression levels measured by flow cytometry. Empty beads were included as a negative control (ctr). Data are presented as the mean ± SD of four separate experiments. Statistical analysis performed using the Friedman one‐way ANOVA test indicated no statistical difference between the different culture conditions. (B) Western blot analysis using 40 µg of indicated EV isolates. Representative of three independent experiments. (C) Measurement of apoptosis in HCT 116 cancer cells after 36 h as detected with Caspase 3/7 green detection reagent, using 40 µg purified EVs from the indicated NK‐92 culture conditions. Data are presented as the mean ± SD of four separate experiments. Statistical analysis performed using the Friedman one‐way ANOVA test indicated no statistical difference between the different culture conditions.

Article Snippet: After transfer onto PVDF membranes (ThermoFisher Scientific), membranes were blocked with 5% dry milk and incubated overnight at 4°C with primary antibodies against CD63 (Ts63, 1:500) and CD81 (M38, 1:500) from ThermoFisher Scientific, and against granzyme B (#2103A, 1:500) from R&D Systems.

Techniques: Functional Assay, Staining, Control, Expressing, Flow Cytometry, Negative Control, Western Blot, Purification

Journal: Biology

Article Title: Development of Liver-Targeting α V β 5 + Exosomes as Anti-TGF-β Nanocarriers for the Treatment of the Pre-Metastatic Niche

doi: 10.3390/biology13121066

Figure Lengend Snippet: Integrin expression in 293T cells and validation of isolated exosomes. ( A ) Expression of the integrins α v ( left ), β 5 ( middle ), and α v β 5 ( right ) in 293T cells using flow cytometry. Results are presented as histograms. ( B ) Western blot analysis of the exosomal marker CD81 in whole cell lysates (WCL) and exosomes (Exo) from 293T cells. ( C ) Measurement of the hydrodynamic diameter of the exosomes isolated from 293T cells. Results are presented as a histogram of the particle size distribution. ( D ) Characterization of the exosome morphology and size using TEM. Results are presented as a representative image ( left ; arrows indicate the exosomes) and a histogram ( right ) of the exosome size distribution.

Article Snippet: Membranes were then incubated overnight at 16 °C in the blocking solution (TBS-T-Milk) containing a primary antibody against CD81 (dilution 1:250 or 0.8 μg/mL, mouse monoclonal IgG 2b κ, clone B-11, Santa Cruz Biotechnology, Dallas, TX, USA), T7 (dilution 1:2500 or 0.4 μg/mL, rabbit polyclonal, Merck Millipore), HA (dilution 1:200 or 1.0 μg/mL, mouse monoclonal, IgG 2a κ, clone F-7, Santa Cruz Biotechnology), Myc (dilution 1:1000 or 0.2 μg/mL, mouse monoclonal IgG 1 , clone 9E10, Santa Cruz Biotechnology), phospho-SMAD2/3 (dilution 1:1000, rabbit polyclonal, Cell Signaling, Danvers, MA, USA), SMAD2/3 (dilution 1:1000, rabbit polyclonal, Cell Signaling), or α-tubulin (dilution 1:8000 or 0.625 μg/mL, mouse monoclonal IgG 1 κ, clone B-5-1-2, Sigma-Aldrich, St. Louis, MO, USA).

Techniques: Expressing, Biomarker Discovery, Isolation, Flow Cytometry, Western Blot, Marker

Journal: Biology

Article Title: Development of Liver-Targeting α V β 5 + Exosomes as Anti-TGF-β Nanocarriers for the Treatment of the Pre-Metastatic Niche

doi: 10.3390/biology13121066

Figure Lengend Snippet: α v β 5 + exosomes derived from 293T-α v β 5 + cells carry the exogenous integrin and are internalized in RAW 264.7 macrophages in vitro. ( A ) Western blot analysis of the levels of the exogenous integrin subunit ITGB5-T7 in exosomes isolated from parental 293T cells (Exo-Prtl) and from 293T-α v β 5 + cells (Exo-α v β 5 ). CD81 was used as a loading control. ( B ) Measurement of the hydrodynamic diameter of the exosomes isolated from 293T cells, either parental (Exo-Prtl) or α v β 5 + (Exo-α v β 5 ). Results are presented as a histogram of the particle size distribution. ( C ) Fluorescence microscopy of RAW 264.7 cells incubated for 24 h with a control solution (PBS SP-DiIC ) or SP-DiIC 18 -stained exosomes (3.0 µg/cm 2 , red) isolated from parental 293T cells (Exo-Prtl SP-DiIC ) and from 293T-α v β 5 + cells (Exo-α v β 5 SP-DiIC ) (1.0 µM SP-DiIC 18 ). Nuclei were stained with DAPI (blue). ( D ) RAW 264.7 cells were cultured for 24 h in the presence or absence of Exo-Prtl or Exo-α v β 5 exosomes (1.5–6.0 µg/cm 2 ) stained with SP-DiIC 18 (1.0 µM) before analyzing cells by flow cytometry. Results are presented as the percentage of exosome-positive RAW 264.7 cells (upper bar graph) and the average ± SEM of the mean fluorescence intensity (MFI, a.u., bottom bar graph). ( E ) Western blot analysis of T7 tag (ITGB5) levels in cell lysates of RAW 264.7 cells incubated for 24 h with PBS, Exo-Prtl exosomes, or Exo-α v β 5 exosomes (3.0 µg/cm 2 ). α-tubulin was used as a loading control.

Article Snippet: Membranes were then incubated overnight at 16 °C in the blocking solution (TBS-T-Milk) containing a primary antibody against CD81 (dilution 1:250 or 0.8 μg/mL, mouse monoclonal IgG 2b κ, clone B-11, Santa Cruz Biotechnology, Dallas, TX, USA), T7 (dilution 1:2500 or 0.4 μg/mL, rabbit polyclonal, Merck Millipore), HA (dilution 1:200 or 1.0 μg/mL, mouse monoclonal, IgG 2a κ, clone F-7, Santa Cruz Biotechnology), Myc (dilution 1:1000 or 0.2 μg/mL, mouse monoclonal IgG 1 , clone 9E10, Santa Cruz Biotechnology), phospho-SMAD2/3 (dilution 1:1000, rabbit polyclonal, Cell Signaling, Danvers, MA, USA), SMAD2/3 (dilution 1:1000, rabbit polyclonal, Cell Signaling), or α-tubulin (dilution 1:8000 or 0.625 μg/mL, mouse monoclonal IgG 1 κ, clone B-5-1-2, Sigma-Aldrich, St. Louis, MO, USA).

Techniques: Derivative Assay, In Vitro, Western Blot, Isolation, Control, Fluorescence, Microscopy, Incubation, Staining, Cell Culture, Flow Cytometry