IRUS Total

Investigation of microvesicle uptake by mouse lung-marginated monocytes in vitro.

File Description SizeFormat 
Bio-protocol4307.pdfPublished version2.71 MBAdobe PDFView/Open
Title: Investigation of microvesicle uptake by mouse lung-marginated monocytes in vitro.
Authors: Tan, YY
O'Dea, KP
Patel, BV
Takata, M
Item Type: Journal Article
Abstract: 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.
Issue Date: 5-Feb-2022
Date of Acceptance: 29-Nov-2021
URI: http://hdl.handle.net/10044/1/95976
DOI: 10.21769/BioProtoc.4307
ISSN: 2331-8325
Publisher: Bio-Protocol
Start Page: 1
End Page: 1
Journal / Book Title: Bio-protocol
Volume: 12
Issue: 3
Copyright Statement: © 2022 The Authors; exclusive licensee Bio-protocal LLC.
Sponsor/Funder: CW+
Funder's Grant Number: Award 0096
Keywords: Extracellular vesicles
Extracellular vesicles
Publication Status: Published
Conference Place: United States
Online Publication Date: 2022-02-05
Appears in Collections:Department of Surgery and Cancer