Detecting individual extracellular vesicles using a multicolor in situ proximity ligation assay with flow cytometric readout.
Flow cytometry is a powerful method for quantitative and qualitative analysis of individual cells. However, flow cytometric analysis of extracellular vesicles (EVs), and the proteins present on their surfaces has been hampered by the small size of the EVs - in particular for the smallest EVs, which can be as little as 40 nm in diameter, the limited number of antigens present, and their low refractive index. We addressed these limitations for detection and characterization of EV by flow cytometry through the use of multiplex and multicolor in situ proximity ligation assays (in situ PLA), allowing each detected EV to be easily recorded over background noise using a conventional flow cytometer. By targeting sets of proteins on the surface that are specific for distinct classes of EVs, the method allows for selective recognition of populations of EVs in samples containing more than one type of EVs. The method presented herein opens up for analyses of EVs using flow cytometry for their characterization and quantification.
Department of Immunology, Genetics &Pathology, Science for Life Laboratory, Uppsala University, SE-751 08 Uppsala, Sweden.
Liza
L
Löf
Department of Immunology, Genetics &Pathology, Science for Life Laboratory, Uppsala University, SE-751 08 Uppsala, Sweden.
Tonge
T
Ebai
Department of Medical Sciences, Clinical Chemistry, Uppsala University, SE-751 85 Uppsala, Sweden.
Louise
L
Dubois
Department of Immunology, Genetics &Pathology, Science for Life Laboratory, Uppsala University, SE-751 08 Uppsala, Sweden.
Lotta
L
Wik
Department of Medical Sciences, Clinical Chemistry, Uppsala University, SE-751 85 Uppsala, Sweden.
K Göran
KG
Ronquist
Department of Immunology, Genetics &Pathology, Science for Life Laboratory, Uppsala University, SE-751 08 Uppsala, Sweden.
Olivia
O
Nolander
Department of Immunology, Genetics &Pathology, Science for Life Laboratory, Uppsala University, SE-751 08 Uppsala, Sweden.
Emma
E
Lundin
Department of Immunology, Genetics &Pathology, Science for Life Laboratory, Uppsala University, SE-751 08 Uppsala, Sweden.
Ola
O
Söderberg
Department of Immunology, Genetics &Pathology, Science for Life Laboratory, Uppsala University, SE-751 08 Uppsala, Sweden.
Ulf
U
Landegren
Department of Immunology, Genetics &Pathology, Science for Life Laboratory, Uppsala University, SE-751 08 Uppsala, Sweden.
Masood
M
Kamali-Moghaddam
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