Extracellular Vesicle Protein Analysis

EV proteins were extracted and analyzed by western blotting as previously reported (Choi et al., 2020 (link)) with few modifications as detailed in Supplementary Materials. EV surface marker analysis was performed by MACSPlex Exosome kit (Miltenyi Biotech; no. 130‐108‐813) following the manufacturer's protocol tube for overnight capture on tubes. HER2 protein detection on EV was performed by staining samples with HER2/ErbB2 (29D8)—PE‐conjugated antibody or Isotype Control (Cell Signaling Technology) and CellMask plasma membrane stains (C10046, Life Technologies). HER2‐positive EV were identified on an Amnis ImageStream X MkII (Luminex).

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Mugoni V., Ciani Y., Quaini O., Tomasini S., Notarangelo M., Vannuccini F., Marinelli A., Leonardi E., Pontalti S., Martinelli A., Rossetto D., Pesce I., Mansy S.S., Barbareschi M., Ferro A., Caffo O., Attard G., Di Vizio D., D'Agostino V.G., Nardella C, & Demichelis F. (2023). Integrating extracellular vesicle and circulating cell‐free DNA analysis using a single plasma aliquot improves the detection of HER2 positivity in breast cancer patients. Journal of Extracellular Biology, 2(9), e108.

Publication 2023

MACSPlex Exosome kit Isotype Control CellMask plasma membrane stains

Corresponding Organization : University of Trento

Other organizations : Ospedale Santa Chiara, University College London, London Cancer, Cedars-Sinai Medical Center

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Variable analysis

independent variables

EV proteins extracted and analyzed by western blotting as previously reported (Choi et al., 2020) with few modifications as detailed in Supplementary Materials
EV surface marker analysis performed by MACSPlex Exosome kit (Miltenyi Biotech; no. 130‐108‐813) following the manufacturer's protocol tube for overnight capture on tubes
HER2 protein detection on EV performed by staining samples with HER2/ErbB2 (29D8)—PE‐conjugated antibody or Isotype Control (Cell Signaling Technology) and CellMask plasma membrane stains (C10046, Life Technologies)

dependent variables

HER2-positive EV identified on an Amnis ImageStream X MkII (Luminex)

control variables

Isotype Control (Cell Signaling Technology)

positive controls

Isotype Control (Cell Signaling Technology)

negative controls

Not explicitly mentioned

Annotations

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