Journal: Journal of Extracellular Biology

Article Title: A One‐Step Workflow for Size‐Based Separation of Extracellular Vesicles With Integrated Surface Marker Detection

doi: 10.1002/jex2.70109

Figure Lengend Snippet: Representation and validation of the AF4‐MALS‐FLD method . (A) Overview of the workflow used for identification of EV surface proteins. PE‐conjugated antibodies were incubated with the sample (e.g. pre‐purified EVs, cell culture supernatant, urine, or plasma) and loaded into the AF4 channel. (B) The light scatter elution profile (in relative scale) (black, full line), UV elution profile (black, dotted line) and the size determination ( R rms in nm) (red) obtained by the multi‐angle light scattering (MALS) detector is plotted against time for labelling of SK‐BR‐3‐derived EVs with PE‐conjugated anti‐CD81 antibody. (C) The fluorescent light detector (FLD) signal (in relative scale) for SK‐BR‐3‐derived EVs labelled with PE‐conjugated anti‐CD9, anti‐CD63 and anti‐CD81 is plotted against time. (D) Transmission electron microscopy (TEM) images of different fractions of the AF4‐MALS‐FLD elution profile are shown (scale bar = 200 nm).

Article Snippet: The following primary and secondary antibodies were used for western blot analysis: mouse monoclonal anti‐Alix (1:1000) (cat no. 2171S, Cell Signaling Technology), rabbit monoclonal anti‐CD9 (1:1000) (cat no. 13403S, Cell Signaling Technology), rabbit monoclonal anti‐Syntenin‐1 (1:1000) (cat no. ab133267, Abcam), mouse monoclonal anti‐TSG101 (1:1000) (cat no. sc‐7964, Santa Cruz Biotechnology), rabbit monoclonal anti‐PSMA (1:1000) (cat no. 12702S), mouse monoclonal anti‐EpCAM (1:1000) (cat no. 2929S, Cell Signaling Technology), rabbit monoclonal anti‐HER2 (1:1000) (cat no. 2165S, Cell Signaling Technology), mouse monoclonal anti‐GAPDH (1:2500) (cat no. G8795, Merck Life Science), sheep anti‐mouse horseradish peroxidase‐linked (1:3000) (cat no. NA931V, GE Healthcare Life Sciences) and donkey anti‐rabbit horseradish peroxidase‐linked antibody (1:8000) (cat no. NA934V, GE Healthcare Life Sciences).

Techniques: Biomarker Discovery, Incubation, Purification, Cell Culture, Clinical Proteomics, Multi-Angle Light Scattering, Derivative Assay, Transmission Assay, Electron Microscopy

Journal: Journal of Extracellular Biology

Article Title: A One‐Step Workflow for Size‐Based Separation of Extracellular Vesicles With Integrated Surface Marker Detection

doi: 10.1002/jex2.70109

Figure Lengend Snippet: AF4‐MALS‐FLD analysis of EV surface proteins with biomarker potential in prostate and breast cancer . MCF‐7‐, MDA‐MB‐231‐ and SK‐BR‐3‐derived EVs were labelled with PE‐conjugated anti‐EpCAM antibodies and analysed by AF4‐MALS‐FLD. (A) The elution profile (in relative scale) of the multi‐angle light scatter (MALS) detector and the size ( R rms in nm) were plotted against time. The fluorescent light detector (FLD) signal for MCF‐7‐, MDA‐MB‐231‐ and SK‐BR‐3‐derived EVs labelled with (B) PE‐conjugated anti‐EpCAM and (C) PE‐conjugated anti‐HER2 antibodies were plotted. (D) From FLD elution profiles, the area under the curve for the EV peak (24–80 min) was determined. Unstained EV samples were used as a negative control. (E) Different concentrations (6 × 10 9 , 8 × 10 9 , 1 × 10 10 and 2 × 10 10 particles as measured by NTA) including a negative control of LNCaP‐derived EVs (high PSMA expression) were labelled with anti‐PSMA antibodies and analysed by the AF4‐MALS‐FLD protocol. (F) The area under the curve for the EV peak was determined for LNCaP‐derived EVs. Different concentrations (2 × 10 10 , 4 × 10 10 and 6 × 10 10 particles as measured by NTA) including a negative control of (G) MCF‐7‐derived EVs (high EpCAM expression) or (I) SK‐BR‐3‐derived EVs (high HER2 expression) were labelled with PE‐conjugated anti‐EpCAM or anti‐HER2 antibodies respectively and analysed by the AF4‐MALS‐FLD protocol. The area under the curve for the EV peak (24–80 min) was determined for (H) MCF‐7‐ and (J) SK‐BR‐3‐derived EVs.

Article Snippet: The following primary and secondary antibodies were used for western blot analysis: mouse monoclonal anti‐Alix (1:1000) (cat no. 2171S, Cell Signaling Technology), rabbit monoclonal anti‐CD9 (1:1000) (cat no. 13403S, Cell Signaling Technology), rabbit monoclonal anti‐Syntenin‐1 (1:1000) (cat no. ab133267, Abcam), mouse monoclonal anti‐TSG101 (1:1000) (cat no. sc‐7964, Santa Cruz Biotechnology), rabbit monoclonal anti‐PSMA (1:1000) (cat no. 12702S), mouse monoclonal anti‐EpCAM (1:1000) (cat no. 2929S, Cell Signaling Technology), rabbit monoclonal anti‐HER2 (1:1000) (cat no. 2165S, Cell Signaling Technology), mouse monoclonal anti‐GAPDH (1:2500) (cat no. G8795, Merck Life Science), sheep anti‐mouse horseradish peroxidase‐linked (1:3000) (cat no. NA931V, GE Healthcare Life Sciences) and donkey anti‐rabbit horseradish peroxidase‐linked antibody (1:8000) (cat no. NA934V, GE Healthcare Life Sciences).

Techniques: Biomarker Discovery, Derivative Assay, Multi-Angle Light Scattering, Negative Control, Expressing

Journal: Journal of Extracellular Biology

Article Title: A One‐Step Workflow for Size‐Based Separation of Extracellular Vesicles With Integrated Surface Marker Detection

doi: 10.1002/jex2.70109

Figure Lengend Snippet: Detection of EVs in complex matrices . (A) Different volumes of cell culture supernatant (0, 20, 40 and 60 µL) collected from the MCF‐7 cells were labelled with PE‐conjugated anti‐EpCAM antibodies and analysed by AF4‐MALS‐FLD. The area under the curve for the EV peak in complex matrices (40–80 min) was determined. (B) Different amounts of LNCaP‐derived EVs were spiked in 100 µL of concentrated urine, diluted 1:1 in PBS to reduce viscosity, labelled with PE‐conjugated anti‐PSMA antibodies, and analysed by AF4‐MALS‐FLD. The area under the curve for the EV peak was determined. Different amounts of (C) MCF‐7‐ or (D) SK‐BR‐3‐derived EVs were spiked in 100 µL of blood plasma, diluted 1:1 in PBS to reduce viscosity, and labelled with PE‐conjugated anti‐EpCAM or anti‐HER2 antibodies, respectively. Labelled EVs were analysed by AF4‐MALS‐FLD and the area under the curve for the EV peak was determined. Different amounts of SK‐BR‐3 EVs were also spiked in blood plasma and labelled with isotype control antibodies. (E) Different concentrations of soluble EpCAM (1, 5 and 10 ng/mL) and soluble HER2 (50, 100 and 150 ng/mL) were spiked in blood plasma, labelled with PE‐conjugated anti‐EpCAM or anti‐HER2 antibodies respectively, and analysed by AF4‐MALS‐FLD.

Article Snippet: The following primary and secondary antibodies were used for western blot analysis: mouse monoclonal anti‐Alix (1:1000) (cat no. 2171S, Cell Signaling Technology), rabbit monoclonal anti‐CD9 (1:1000) (cat no. 13403S, Cell Signaling Technology), rabbit monoclonal anti‐Syntenin‐1 (1:1000) (cat no. ab133267, Abcam), mouse monoclonal anti‐TSG101 (1:1000) (cat no. sc‐7964, Santa Cruz Biotechnology), rabbit monoclonal anti‐PSMA (1:1000) (cat no. 12702S), mouse monoclonal anti‐EpCAM (1:1000) (cat no. 2929S, Cell Signaling Technology), rabbit monoclonal anti‐HER2 (1:1000) (cat no. 2165S, Cell Signaling Technology), mouse monoclonal anti‐GAPDH (1:2500) (cat no. G8795, Merck Life Science), sheep anti‐mouse horseradish peroxidase‐linked (1:3000) (cat no. NA931V, GE Healthcare Life Sciences) and donkey anti‐rabbit horseradish peroxidase‐linked antibody (1:8000) (cat no. NA934V, GE Healthcare Life Sciences).

Techniques: Cell Culture, Derivative Assay, Viscosity, Clinical Proteomics, Control

Journal: Journal of Extracellular Biology

Article Title: A One‐Step Workflow for Size‐Based Separation of Extracellular Vesicles With Integrated Surface Marker Detection

doi: 10.1002/jex2.70109

Figure Lengend Snippet: Validation of the AF4‐MALS‐FLD workflow on patient samples . Urine samples of five prostate cancer patients were labelled for PSMA and analysed by the AF4‐MALS‐FLD workflow. Fractions 40–80 min were collected, concentrated and processed for mass spectrometry‐based proteomic analysis. (A) EV markers Syntenin‐1, Flotillin‐1, CD63, CD9, CD81, Flotillin‐2, Alix and TSG101 were analysed (missing sample indicated in grey). Z ‐score transformation of intensities were plotted. (B) Targeted mass spectrometry analysed the presence of PSMA (FOLH1) in patient samples. The z ‐score transformation of intensities was plotted with the AF4‐MALS‐FLD peak area. (C) Blood plasma samples of healthy controls ( n = 7) and HER2 amplified breast cancer patients ( n = 10) were labelled with PE‐conjugated anti‐HER2 antibodies. (D) Blood plasma samples of healthy controls ( n = 6) and breast cancer patients ( n = 8) were labelled with PE‐conjugated anti‐EpCAM antibodies. The area under the curve values were normalised for the mean value in the healthy control group.

Article Snippet: The following primary and secondary antibodies were used for western blot analysis: mouse monoclonal anti‐Alix (1:1000) (cat no. 2171S, Cell Signaling Technology), rabbit monoclonal anti‐CD9 (1:1000) (cat no. 13403S, Cell Signaling Technology), rabbit monoclonal anti‐Syntenin‐1 (1:1000) (cat no. ab133267, Abcam), mouse monoclonal anti‐TSG101 (1:1000) (cat no. sc‐7964, Santa Cruz Biotechnology), rabbit monoclonal anti‐PSMA (1:1000) (cat no. 12702S), mouse monoclonal anti‐EpCAM (1:1000) (cat no. 2929S, Cell Signaling Technology), rabbit monoclonal anti‐HER2 (1:1000) (cat no. 2165S, Cell Signaling Technology), mouse monoclonal anti‐GAPDH (1:2500) (cat no. G8795, Merck Life Science), sheep anti‐mouse horseradish peroxidase‐linked (1:3000) (cat no. NA931V, GE Healthcare Life Sciences) and donkey anti‐rabbit horseradish peroxidase‐linked antibody (1:8000) (cat no. NA934V, GE Healthcare Life Sciences).

Techniques: Biomarker Discovery, Mass Spectrometry, Transformation Assay, Clinical Proteomics, Amplification, Control