Imperial College London
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DrKieranO'Dea

Faculty of Medicine, Department of Surgery & Cancer

Senior Lecturer in Translational Critical Care
 
 
 
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Contact

 

k.odea

 
 
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Location

 

G3.43Chelsea and Westminster HospitalChelsea and Westminster Campus

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Summary

 

Publications

Citation

BibTex format

@article{Tan:2022:10.21769/BioProtoc.4307,
author = {Tan, YY and O'Dea, KP and Patel, BV and Takata, M},
doi = {10.21769/BioProtoc.4307},
journal = {Bio-protocol},
pages = {1--1},
title = {Investigation of microvesicle uptake by mouse lung-marginated monocytes in vitro.},
url = {http://dx.doi.org/10.21769/BioProtoc.4307},
volume = {12},
year = {2022}
}
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RIS format (EndNote, RefMan)

TY  - JOUR
AB  - Extracellular microvesicles (MVs) are released into the circulation in large numbers during acute systemic inflammation, yet little is known of their intravascular cell/tissue-specific interactions under these conditions. We recently described a dramatic increase in the uptake of intravenously injected MVs by monocytes marginated within the pulmonary vasculature, in a mouse model of low-dose lipopolysaccharide-induced systemic inflammation. To investigate the mechanisms of enhanced MV uptake by monocytes, we developed an in vitro model using in vivo derived monocytes. Although mouse blood is a convenient source, monocyte numbers are too low for in vitro experimentation. In contrast, differentiated bone marrow monocytes are abundant, but they are rapidly mobilized during systemic inflammation, and thus no longer available. Instead, we developed a protocol using marginated monocytes from the pulmonary vasculature as an anatomically relevant and abundant source. Mice are sacrificed by terminal anesthesia, the lungs inflated and perfused via the pulmonary artery. Perfusate cell populations are evaluated by flow cytometry, combined with in vitro generated fluorescently labelled MVs, and incubated in suspension for up to one hour. Washed cells are analyzed by flow cytometry to quantify MV uptake and confocal microscopy to localize MVs within cells (O'Dea et al., 2020). Using this perfusion-based method, substantial numbers of marginated pulmonary vascular monocytes are recovered, allowing multiple in vitro tests to be performed from a single mouse donor. As MV uptake profiles were comparable to those observed in vivo, this method is suitable for physiologically relevant high throughput mechanistic studies on mouse monocytes under in vitro conditions. Graphic abstract: Figure 1. Schematic of lung perfusate cell harvest and co-incubation with in vitro generated MVs. Created with BioRender.com.
AU  - Tan,YY
AU  - O'Dea,KP
AU  - Patel,BV
AU  - Takata,M
DO  - 10.21769/BioProtoc.4307
EP  - 1
PY  - 2022///
SN  - 2331-8325
SP  - 1
TI  - Investigation of microvesicle uptake by mouse lung-marginated monocytes in vitro.
T2  - Bio-protocol
UR  - http://dx.doi.org/10.21769/BioProtoc.4307
UR  - https://www.ncbi.nlm.nih.gov/pubmed/35284602
UR  - https://bio-protocol.org/e4307
UR  - http://hdl.handle.net/10044/1/95976
VL  - 12
ER  -
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