Monitoring Cellular Proliferation, Migration, and Apoptosis Associated with Atherosclerosis Plaques In Vitro

Kerry S Wadey, Alexandros Somos, Stephen J Cross, Lien M Reolizo, Jason L Johnson, Sarah J George

Research output: Chapter in Book/Report/Conference proceedingChapter in a book

2 Citations (Scopus)


Bromodeoxyuridine/5-bromo-2'-deoxyuridine (BrdU) is a nucleoside analog of thymidine and its incorporation into DNA during replication within S-phase of the cell cycle is used to quantify cell proliferation. Quantification of incorporated BrdU is considered the most direct measure of cell proliferation, and here we describe BrdU incorporation into cultured vascular smooth muscle cells (VSMCs) and endothelial cells in vitro. Incorporation of fluorescent-labeled ethynyldeoxyuridine/5-ethynyl-2'-deoxyuridine (EdU) is a novel alternative to BrdU assays and presents significant advantages. This method of detection of EdU based on a simple "click" chemical reaction, which covalently bonds EdU to a fluorescent dye is also outlined in this chapter with a protocol for quantitative analysis of EdU incorporation using a Fiji-based macro. We also describe how proliferation can be assessed by quantification of classical proliferative markers such as phopsho-Ser807/811 retinoblastoma (Rb), proliferating cell nuclear antigen (PCNA) and cyclin D1 by Western blotting. As these markers are involved in different aspects of the cell cycle regulation, examining their expression levels can not only reveal the relative population of proliferating cells but can also improve our understanding of the mechanism of action of a given treatment or intervention. The scratch wound assay is a simple and cost-effective technique to quantify cell migration. A protocol which involves creating a wound in a cell cultured monolayer and measuring the distance migrated by the cells after a predefined time period is also described. Gap creation can also be achieved via physical cell exclusion where cells are seeded in distinct reservoirs of a cell culture insert which reveal a gap upon removal. Cell migration may then be quantified by monitoring the rate of gap closure. The presence of cleaved caspase-3 is a marker of programmed cell death (apoptosis). To detect cleaved caspase-3 in vitro, immunocytochemistry and fluorescence can be performed as outlined in this chapter.

Original languageEnglish
Title of host publicationMethods in Molecular Biology
EditorsDipak Ramji
Number of pages35
Publication statusPublished - Feb 2022

Publication series

NameMethods in Molecular Biology
PublisherHumana Press
ISSN (Print)1064-3745

Bibliographical note

Funding Information:
Alexandros Somos is funded by a British Heart Foundation PhD studentship, FS/18/74/33976. Jason Johnson is funded by British Heart Foundation Senior Research Fellowship, FS/18/1/ 33234.

Publisher Copyright:
© 2022, The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.


  • Apoptosis
  • Atherosclerosis
  • Bromodeoxyuridine/metabolism
  • Cell Proliferation
  • Deoxyuridine
  • Endothelial Cells/metabolism
  • Humans


Dive into the research topics of 'Monitoring Cellular Proliferation, Migration, and Apoptosis Associated with Atherosclerosis Plaques In Vitro'. Together they form a unique fingerprint.

Cite this