An adapted novel flow cytometry methodology to delineate types of cell death in airway epithelial cells
Main Article Content
Keywords
airway epithelium, cell death, rhinovirus, flow cytometry
Abstract
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.
Downloads
Download data is not yet available.
Metrics
Metrics Loading ...
References
1. Stick SM, Brennan S, Murray C, Douglas T, von Ungern-Sternberg BS, Garratt LW, et al. Bronchiectasis in infants and preschool children diagnosed with cystic fibrosis after newborn screening. J Pediatr. 2009;155(5):623-8 e1.
2. Deschamp AR, Hatch JE, Slaven JE, Gebregziabher N, Storch G, Hall GL, et al. Early respiratory viral infections in infants with cystic fibrosis. J Cyst Fibros. 2019.
3. van Ewijk BE, van der Zalm MM, Wolfs TF, Fleer A, Kimpen JL, Wilbrink B, et al. Prevalence and impact of respiratory viral infections in young children with cystic fibrosis: prospective cohort study. Pediatrics. 2008;122(6):1171-6.
4. Gern JE, Dick EC, Lee WM, Murray S, Meyer K, Handzel ZT, et al. Rhinovirus enters but does not replicate inside monocytes and airway macrophages. J Immunol. 1996;156(2):621-7.
5. Sutanto EN, Kicic A, Foo CJ, Stevens PT, Mullane D, Knight DA, et al. Innate inflammatory responses of pediatric cystic fibrosis airway epithelial cells: effects of nonviral and viral stimulation. Am J Respir Cell Mol Biol. 2011;44(6):761-7.
6. Lötzerich M, Roulin PS, Boucke K, Witte R, Georgiev O, Greber UF. Rhinovirus 3C protease suppresses apoptosis and triggers caspase-independent cell death. Cell Death Dis. 2018;9(3):272.
7. Fritzsching B, Zhou-Suckow Z, Trojanek JB, Schubert SC, Schatterny J, Hirtz S, et al. Hypoxic epithelial necrosis triggers neutrophilic inflammation via IL-1 receptor signaling in cystic fibrosis lung disease. Am J Respir Crit Care Med. 2015;191(8):902-13.
8. Montgomery ST, Dittrich AS, Garratt LW, Turkovic L, Frey DL, Stick SM, et al. Interleukin-1 is associated with inflammation and structural lung disease in young children with cystic fibrosis. J Cyst Fibros. 2018;17(6):715-22.
9. Vermes I, Haanen C, Steffens-Nakken H, Reutellingsperger C. A novel assay for apoptosis Flow cytometric detection of phosphatidylserine expression on early apoptotic cells using fluorescein labelled Annexin V. J Immunol Methods. 1995;184(1):39-51.
10. Faleiro L, Lazebnik Y. Caspases Disrupt the Nuclear-Cytoplasmic Barrier. The Journal of Cell Biology. 2000;151(5):951.
11. Berghe TV, Vanlangenakker N, Parthoens E, Deckers W, Devos M, Festjens N, et al. Necroptosis, necrosis and secondary necrosis converge on similar cellular disintegration features. Cell Death Differ. 2009;17:922.
12. Rieger AM, Hall BE, Luong LT, Schang LM, Barreda DR. Conventional apoptosis assays using propidium iodide generate a significant number of false positives that prevent accurate assessment of cell death. J Immunol Methods. 2010;358(1):81-92.
13. Sawai H, Domae N. Discrimination between primary necrosis and apoptosis by necrostatin-1 in Annexin V-positive/propidium iodide-negative cells. Biochem Biophys Res Commun. 2011;411(3):569-73.
14. Jiang L, Tixeira R, Caruso S, Atkin-Smith GK, Baxter AA, Paone S, et al. Monitoring the progression of cell death and the disassembly of dying cells by flow cytometry. Nat Protoc. 2016;11:655.
15. Poon IKH, Chiu Y-H, Armstrong AJ, Kinchen JM, Juncadella IJ, Bayliss DA, et al. Unexpected link between an antibiotic, pannexin channels and apoptosis. Nature. 2014;507(7492):329-34.
16. Orlando KA, Stone NL, Pittman RN. Rho kinase regulates fragmentation and phagocytosis of apoptotic cells. Exp Cell Res. 2006;312(1):5-15.
17. Berda-Haddad Y, Robert S, Salers P, Zekraoui L, Farnarier C, Dinarello CA, et al. Sterile inflammation of endothelial cell-derived apoptotic bodies is mediated by interleukin-1alpha. Proc Natl Acad Sci U S A. 2011;108(51):20684-9.
18. Zernecke A, Bidzhekov K, Noels H, Shagdarsuren E, Gan L, Denecke B, et al. Delivery of microRNA-126 by apoptotic bodies induces CXCL12-dependent vascular protection. Sci Signal. 2009;2(100):ra81.
19. Martinovich KM, Iosifidis T, Buckley AG, Looi K, Ling K-M, Sutanto EN, et al. Conditionally reprogrammed primary airway epithelial cells maintain morphology, lineage and disease specific functional characteristics. Scientific Reports. 2017;7(1):17971.
20. Lee W-M, Chen Y, Wang W, Mosser A. Growth of Human Rhinovirus in H1-HeLa Cell Suspension Culture and Purification of Virions. In: Jans DA, Ghildyal R, editors. Rhinoviruses: Methods and Protocols. New York, NY: Springer New York; 2015. p. 49-61.
21. Bochkov YA, Palmenberg AC, Lee W-M, Rathe JA, Amineva SP, Sun X, et al. Molecular modeling, organ culture and reverse genetics for a newly identified human rhinovirus C. Nat Med. 2011;17(5):627-32.
22. Vandivier RW, Henson PM, Douglas IS. Burying the dead: the impact of failed apoptotic cell removal (efferocytosis) on chronic inflammatory lung disease. Chest. 2006;129(6):1673-82.
23. Szondy Z, Sarang Z, Kiss B, Garabuczi É, Köröskényi K. Anti-inflammatory Mechanisms Triggered by Apoptotic Cells during Their Clearance. Frontiers in immunology. 2017;8:909-.
24. Silva MT, Correia-Neves M. Neutrophils and macrophages: the main partners of phagocyte cell systems. Frontiers in immunology. 2012;3:174.
25. Juncadella IJ, Kadl A, Sharma AK, Shim YM, Hochreiter-Hufford A, Borish L, et al. Apoptotic cell clearance by bronchial epithelial cells critically influences airway inflammation. Nature. 2013;493(7433):547-51.
26. Gray RD, Hardisty G, Regan KH, Smith M, Robb CT, Duffin R, et al. Delayed neutrophil apoptosis enhances NET formation in cystic fibrosis. Thorax. 2018;73(2):134-44.
27. Vandivier RW, Fadok VA, Hoffmann PR, Bratton DL, Penvari C, Brown KK, et al. Elastase-mediated phosphatidylserine receptor cleavage impairs apoptotic cell clearance in cystic fibrosis and bronchiectasis. The Journal of Clinical Investigation. 2002;109(5):661-70.
28. Forrest OA, Ingersoll SA, Preininger MK, Laval J, Limoli DH, Brown MR, et al. Frontline Science: Pathological conditioning of human neutrophils recruited to the airway milieu in cystic fibrosis. J Leukoc Biol. 2018;104(4):665-75.
29. Sachet M, Liang YY, Oehler R. The immune response to secondary necrotic cells. Apoptosis : an international journal on programmed cell death. 2017;22(10):1189-204.
30. Montgomery ST, Frey DL, Mall MA, Stick SM, Kicic A, Cf A. Rhinovirus Infection Is Associated With Airway Epithelial Cell Necrosis and Inflammation via Interleukin-1 in Young Children With Cystic Fibrosis. Frontiers in immunology. 2020;11:596.
2. Deschamp AR, Hatch JE, Slaven JE, Gebregziabher N, Storch G, Hall GL, et al. Early respiratory viral infections in infants with cystic fibrosis. J Cyst Fibros. 2019.
3. van Ewijk BE, van der Zalm MM, Wolfs TF, Fleer A, Kimpen JL, Wilbrink B, et al. Prevalence and impact of respiratory viral infections in young children with cystic fibrosis: prospective cohort study. Pediatrics. 2008;122(6):1171-6.
4. Gern JE, Dick EC, Lee WM, Murray S, Meyer K, Handzel ZT, et al. Rhinovirus enters but does not replicate inside monocytes and airway macrophages. J Immunol. 1996;156(2):621-7.
5. Sutanto EN, Kicic A, Foo CJ, Stevens PT, Mullane D, Knight DA, et al. Innate inflammatory responses of pediatric cystic fibrosis airway epithelial cells: effects of nonviral and viral stimulation. Am J Respir Cell Mol Biol. 2011;44(6):761-7.
6. Lötzerich M, Roulin PS, Boucke K, Witte R, Georgiev O, Greber UF. Rhinovirus 3C protease suppresses apoptosis and triggers caspase-independent cell death. Cell Death Dis. 2018;9(3):272.
7. Fritzsching B, Zhou-Suckow Z, Trojanek JB, Schubert SC, Schatterny J, Hirtz S, et al. Hypoxic epithelial necrosis triggers neutrophilic inflammation via IL-1 receptor signaling in cystic fibrosis lung disease. Am J Respir Crit Care Med. 2015;191(8):902-13.
8. Montgomery ST, Dittrich AS, Garratt LW, Turkovic L, Frey DL, Stick SM, et al. Interleukin-1 is associated with inflammation and structural lung disease in young children with cystic fibrosis. J Cyst Fibros. 2018;17(6):715-22.
9. Vermes I, Haanen C, Steffens-Nakken H, Reutellingsperger C. A novel assay for apoptosis Flow cytometric detection of phosphatidylserine expression on early apoptotic cells using fluorescein labelled Annexin V. J Immunol Methods. 1995;184(1):39-51.
10. Faleiro L, Lazebnik Y. Caspases Disrupt the Nuclear-Cytoplasmic Barrier. The Journal of Cell Biology. 2000;151(5):951.
11. Berghe TV, Vanlangenakker N, Parthoens E, Deckers W, Devos M, Festjens N, et al. Necroptosis, necrosis and secondary necrosis converge on similar cellular disintegration features. Cell Death Differ. 2009;17:922.
12. Rieger AM, Hall BE, Luong LT, Schang LM, Barreda DR. Conventional apoptosis assays using propidium iodide generate a significant number of false positives that prevent accurate assessment of cell death. J Immunol Methods. 2010;358(1):81-92.
13. Sawai H, Domae N. Discrimination between primary necrosis and apoptosis by necrostatin-1 in Annexin V-positive/propidium iodide-negative cells. Biochem Biophys Res Commun. 2011;411(3):569-73.
14. Jiang L, Tixeira R, Caruso S, Atkin-Smith GK, Baxter AA, Paone S, et al. Monitoring the progression of cell death and the disassembly of dying cells by flow cytometry. Nat Protoc. 2016;11:655.
15. Poon IKH, Chiu Y-H, Armstrong AJ, Kinchen JM, Juncadella IJ, Bayliss DA, et al. Unexpected link between an antibiotic, pannexin channels and apoptosis. Nature. 2014;507(7492):329-34.
16. Orlando KA, Stone NL, Pittman RN. Rho kinase regulates fragmentation and phagocytosis of apoptotic cells. Exp Cell Res. 2006;312(1):5-15.
17. Berda-Haddad Y, Robert S, Salers P, Zekraoui L, Farnarier C, Dinarello CA, et al. Sterile inflammation of endothelial cell-derived apoptotic bodies is mediated by interleukin-1alpha. Proc Natl Acad Sci U S A. 2011;108(51):20684-9.
18. Zernecke A, Bidzhekov K, Noels H, Shagdarsuren E, Gan L, Denecke B, et al. Delivery of microRNA-126 by apoptotic bodies induces CXCL12-dependent vascular protection. Sci Signal. 2009;2(100):ra81.
19. Martinovich KM, Iosifidis T, Buckley AG, Looi K, Ling K-M, Sutanto EN, et al. Conditionally reprogrammed primary airway epithelial cells maintain morphology, lineage and disease specific functional characteristics. Scientific Reports. 2017;7(1):17971.
20. Lee W-M, Chen Y, Wang W, Mosser A. Growth of Human Rhinovirus in H1-HeLa Cell Suspension Culture and Purification of Virions. In: Jans DA, Ghildyal R, editors. Rhinoviruses: Methods and Protocols. New York, NY: Springer New York; 2015. p. 49-61.
21. Bochkov YA, Palmenberg AC, Lee W-M, Rathe JA, Amineva SP, Sun X, et al. Molecular modeling, organ culture and reverse genetics for a newly identified human rhinovirus C. Nat Med. 2011;17(5):627-32.
22. Vandivier RW, Henson PM, Douglas IS. Burying the dead: the impact of failed apoptotic cell removal (efferocytosis) on chronic inflammatory lung disease. Chest. 2006;129(6):1673-82.
23. Szondy Z, Sarang Z, Kiss B, Garabuczi É, Köröskényi K. Anti-inflammatory Mechanisms Triggered by Apoptotic Cells during Their Clearance. Frontiers in immunology. 2017;8:909-.
24. Silva MT, Correia-Neves M. Neutrophils and macrophages: the main partners of phagocyte cell systems. Frontiers in immunology. 2012;3:174.
25. Juncadella IJ, Kadl A, Sharma AK, Shim YM, Hochreiter-Hufford A, Borish L, et al. Apoptotic cell clearance by bronchial epithelial cells critically influences airway inflammation. Nature. 2013;493(7433):547-51.
26. Gray RD, Hardisty G, Regan KH, Smith M, Robb CT, Duffin R, et al. Delayed neutrophil apoptosis enhances NET formation in cystic fibrosis. Thorax. 2018;73(2):134-44.
27. Vandivier RW, Fadok VA, Hoffmann PR, Bratton DL, Penvari C, Brown KK, et al. Elastase-mediated phosphatidylserine receptor cleavage impairs apoptotic cell clearance in cystic fibrosis and bronchiectasis. The Journal of Clinical Investigation. 2002;109(5):661-70.
28. Forrest OA, Ingersoll SA, Preininger MK, Laval J, Limoli DH, Brown MR, et al. Frontline Science: Pathological conditioning of human neutrophils recruited to the airway milieu in cystic fibrosis. J Leukoc Biol. 2018;104(4):665-75.
29. Sachet M, Liang YY, Oehler R. The immune response to secondary necrotic cells. Apoptosis : an international journal on programmed cell death. 2017;22(10):1189-204.
30. Montgomery ST, Frey DL, Mall MA, Stick SM, Kicic A, Cf A. Rhinovirus Infection Is Associated With Airway Epithelial Cell Necrosis and Inflammation via Interleukin-1 in Young Children With Cystic Fibrosis. Frontiers in immunology. 2020;11:596.
Article Sidebar
Published
Nov 11, 2020
Article Details
How to Cite
1.
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]. 2020 Nov. 11 [cited 2024 Apr. 26];7(4):e139. Available from: https://jbmethods.org/index.php/jbm/article/view/336
Issue
Section
Articles
Authors who publish with JBM agree to the following terms:
- Authors retain copyright and grant JBM right of first publication with the work simultaneously licensed under a Creative Commons Attribution License that allows others to share the work with an acknowledgement of the work's authorship and initial publication in this journal.
- Authors are able to enter into separate, additional contractual arrangements for the non-exclusive distribution of the journal's published version of the work (e.g., post it to an institutional repository or publish it in a book), with an acknowledgement of its initial publication in this journal.
- Authors are permitted and encouraged to post their work online (e.g., in institutional repositories or on their website) prior to and during the submission process, as it can lead to productive exchanges, as well as earlier and greater citation of published work (See The Effect of Open Access).
