An adapted novel flow cytometry methodology to delineate types of cell death in airway epithelial cells


  • Samuel T. Montgomery Faculty of Medicine and Health Science, University of Western Australia, Western Australia, Australia
  • Stephen M. Stick Faculty of Medicine and Health Science, University of Western Australia, Western Australia, Australia Telethon Kids Institute, University of Western Australia, Western Australia, Australia Department of Respiratory and Sleep Medicine, Perth Children
  • Anthony Kicic Faculty of Medicine and Health Science, University of Western Australia, Western Australia, Australia Telethon Kids Institute, University of Western Australia, Western Australia, Australia Department of Respiratory and Sleep Medicine, Perth Children



airway epithelium, cell death, rhinovirus, flow cytometry


Current methodologies to measure apoptotic and necrotic cell death using flow cytometry do not adequately differentiate between the two. Here, we describe a flow cytometry methodology adapted to airway epithelial cells (AEC) to sufficiently differentiate apoptotic and necrotic AEC. Specifically, cell lines and primary AEC (n = 12) were permeabilized or infected with rhinovirus 1b (RV1b) over 48 h. Cell death was then measured via annexin V/propidium iodide (A5/PI) or annexin V/TO-PRO-3 (A5/TP3) staining using a novel flow cytometry and gating methodology adapted to AEC. We show that A5/PI staining could not sufficiently differentiate between types of cell death following RV1b infection of primary AEC. However, A5/TP3 staining was able to distinguish six cell death populations (viable, necrotic, debris, A5+ apoptotic, A5− apoptotic, apoptotic bodies) after permeabilization or infection with RV1b, with phenotypic differences were observed in apoptotic populations. Collectively, using a staining and gating strategy never adapted to AEC, A5/TP3 could accurately differentiate and quantify viable, necrotic, and apoptotic AEC following RV1b infection.


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How to Cite

Montgomery ST, Stick SM, Kicic A. An adapted novel flow cytometry methodology to delineate types of cell death in airway epithelial cells. J Biol Methods [Internet]. 2020Nov.11 [cited 2021Jul.28];7(4):e139. Available from: