Single-EV Analysis by Imaging Flow Cytometry

A total of 10⁸ EVs were analyzed at the single EV level by imaging flow cytometry (ImageStream X MKII, Amnis/Luminex) following guidelines and principles described in Görgens et al. (28 (link)) (see SI Appendix), with the following labels: lipid dye MemGlow 488 (Cytoskeleton, Inc), anti-CD81-APC (dilution 1/25) (Clone 5A6, Biolegend), and anti-CSF-1-PE (dilution 1/25) (Clone 26786, R&D Systems). Adequate PBS, unstained, Membright488, CD81, CSF-1 monocolors, and fluorescence minus one (FMOs) EVs controls were performed in parallel.

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Tkach M., Thalmensi J., Timperi E., Gueguen P., Névo N., Grisard E., Sirven P., Cocozza F., Gouronnec A., Martin-Jaular L., Jouve M., Delisle F., Manel N., Rookhuizen D.C., Guerin C.L., Soumelis V., Romano E., Segura E, & Théry C. (2022). Extracellular vesicles from triple negative breast cancer promote pro-inflammatory macrophages associated with better clinical outcome. Proceedings of the National Academy of Sciences of the United States of America, 119(17), e2107394119.

Publication 2022

MemGlow 488 anti-CD81-APC

Clone Csf 1 Cytoskeleton Dilution Flow cytometry Fluorescence Lipid

Corresponding Organization :

Other organizations : Université Paris Sciences et Lettres, Inserm, Institut Curie, Centre National de la Recherche Scientifique, Université Paris Cité, Hôpital Saint-Louis, Assistance Publique – Hôpitaux de Paris

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Variable analysis

independent variables

Lipid dye MemGlow 488 (Cytoskeleton, Inc)
Anti-CD81-APC (dilution 1/25) (Clone 5A6, Biolegend)
Anti-CSF-1-PE (dilution 1/25) (Clone 26786, R&D Systems)

dependent variables

Total of 10^8 EVs analyzed at the single EV level by imaging flow cytometry (ImageStream X MKII, Amnis/Luminex)

control variables

Unstained
Membright488
CSF-1 monocolors
Fluorescence minus one (FMOs) EVs controls

positive controls

Membright488
CSF-1 monocolors

negative controls

Unstained
Fluorescence minus one (FMOs) EVs controls

Annotations

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