Phenotyping Extracellular Vesicles by Flow Cytometry

To proceed to further analysis, PPP was first centrifuged at 2000 g for 20 min and thereafter at 13000 g for 2 min at room temperature. As previously described (24 (link), 25 (link)), the supernatant was incubated with monoclonal antibodies in order to phenotype EVs with antibodies against MPO, conjugated with PE (anti-MPO-PE, Beckman Coulter, Brea, CA, United States), tissue factor (CD142D, NJ, United States), citrullinated histone-3 (anti-H3Cit, Abcam, Cambridge, United Kingdom), and plasminogen (Abcam, Cambridge, United Kingdom). The samples were further fixed (Cellfix, BD, NJ, United States) in order to be evaluated by flow cytometry on a Beckman Gallios instrument (Beckman Coulter, Brea, CA, United States). The EVs gate was determined using Megamix beads (0.3–1.0 μm, BioCytex, Marseille). EVs were defined as particles <1 μm in diameter, according to MISEV guidelines defnition of a medium/large EVs (28 (link)). The measured levels of EVs are presented as EVs/μL plasma. The intra- and inter-assay coefficients of variation for MPO⁺EVs measurement were <9%, respectively.

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Jonasdottir A.D., Manojlovic M., Vojinovic J., Nordin A., Bruchfeld A., Gunnarsson I., Mobarrez F, & Antovic A. (2023). Augmented thrombin formation is related to circulating levels of extracellular vesicles exposing tissue factor and citrullinated histone-3 in anti-neutrophil cytoplasmic antibody-associated vasculitides. Frontiers in Medicine, 10, 1240325.

Publication 2023

anti-H3Cit Beckman Gallios Megamix beads

Antibodies Assay Flow cytometry Histone Monoclonal antibodies Phenotype Plasma Plasminogen Tissue factor

Corresponding Organization : Karolinska University Hospital

Other organizations : National University Hospital of Iceland, University of Nis, Linköping University, Uppsala University

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Variable analysis

independent variables

Centrifugation at 2000 g for 20 min
Centrifugation at 13000 g for 2 min

dependent variables

Levels of EVs (extracellular vesicles) expressing MPO, tissue factor, citrullinated histone-3, and plasminogen

control variables

Room temperature
Antibodies against MPO, tissue factor, citrullinated histone-3, and plasminogen

controls

Positive control: Not explicitly mentioned.
Negative control: Not explicitly mentioned.

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