Visualisation of Multiple Tight Junctional Complexes in Human Airway Epithelial Cells

Alysia G. Buckley, Kevin Looi, Thomas Iosifidis, Kak Ming Ling, Erika N. Sutanto, Kelly M. Martinovich, Elizabeth Kicic-Starcevich, Luke W. Garratt, Nicole C. Shaw, Francis J. Lannigan, Alexander N. Larcombe, Graeme Zosky, Darryl A. Knight, Paul J. Rigby, Anthony Kicic, Stephen M. Stick

Research output: Contribution to journalArticle

3 Citations (Scopus)

Abstract

Background: Apically located tight junctions in airway epithelium perform a fundamental role in controlling macromolecule migration through paracellular spaces. Alterations in their expression may lead to disruptions in barrier integrity, which subsequently facilitates entry of potential bacterial and other pathogens into the host. Furthermore, there is emerging evidence that the barrier integrity of the airway in certain airway inflammatory diseases may be altered. However, there is little consensus on the way this is assessed and measured and the type of cells used to achieve this. Methods: Here, we assessed four fixation methods including; (i) 4% (v/v) paraformaldehyde; (ii) 100% methanol; (iii) acetone or; (iv) 1:1 methanol: acetone. Pre-extraction with Triton X-100 was also performed and assessed on cells prior to fixation with either methanol or paraformaldehyde. Cells were also permeabilized with 0.1% (v/v) Saponin in 1× TBS following fixation and subsequently stained for tight junction proteins. Confocal microscopy was then used to visualise, compare and evaluate staining intensity of the tight junctional complexes in order to determine a standardised workflow of reproducible staining. Results: Positive staining was observed following methanol fixation for claudin-1 and ZO-1 tight junction proteins but no staining was detected for occludin in 16HBE14o- cells. Combinatorial fixation with methanol and acetone also produced consistent positive staining for both occludin and ZO-1 tight junction proteins in these cells. When assessed using primary cells cultured at air-liquid interface, similar positive staining for claudin-1 and ZO-1 was observed following methanol fixation, while similar positive staining for occludin and ZO-1 was observed following the same combinatorial fixation with methanol and acetone. Conclusions: The present study demonstrates the importance of a personalised approach to optimise staining for the visualisation of different tight junction proteins. Of significance, the workflow, once optimised, can readily be translated into primary airway epithelial cell air-liquid interface cultures where it can be used to assess barrier integrity in chronic lung diseases.

Original languageEnglish
Article number3
JournalBiological Procedures Online
Volume20
Issue number1
DOIs
Publication statusPublished - 1 Feb 2018

Fingerprint

Methanol
Visualization
Epithelial Cells
Staining and Labeling
Acetone
Occludin
Zonula Occludens-1 Protein
Claudin-1
Tight Junction Proteins
Workflow
Air
Pulmonary diseases
Confocal microscopy
Saponins
Octoxynol
Liquids
Pathogens
Tight Junctions
Macromolecules
Confocal Microscopy

Cite this

Buckley, Alysia G. ; Looi, Kevin ; Iosifidis, Thomas ; Ling, Kak Ming ; Sutanto, Erika N. ; Martinovich, Kelly M. ; Kicic-Starcevich, Elizabeth ; Garratt, Luke W. ; Shaw, Nicole C. ; Lannigan, Francis J. ; Larcombe, Alexander N. ; Zosky, Graeme ; Knight, Darryl A. ; Rigby, Paul J. ; Kicic, Anthony ; Stick, Stephen M. / Visualisation of Multiple Tight Junctional Complexes in Human Airway Epithelial Cells. In: Biological Procedures Online. 2018 ; Vol. 20, No. 1.
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title = "Visualisation of Multiple Tight Junctional Complexes in Human Airway Epithelial Cells",
abstract = "Background: Apically located tight junctions in airway epithelium perform a fundamental role in controlling macromolecule migration through paracellular spaces. Alterations in their expression may lead to disruptions in barrier integrity, which subsequently facilitates entry of potential bacterial and other pathogens into the host. Furthermore, there is emerging evidence that the barrier integrity of the airway in certain airway inflammatory diseases may be altered. However, there is little consensus on the way this is assessed and measured and the type of cells used to achieve this. Methods: Here, we assessed four fixation methods including; (i) 4{\%} (v/v) paraformaldehyde; (ii) 100{\%} methanol; (iii) acetone or; (iv) 1:1 methanol: acetone. Pre-extraction with Triton X-100 was also performed and assessed on cells prior to fixation with either methanol or paraformaldehyde. Cells were also permeabilized with 0.1{\%} (v/v) Saponin in 1× TBS following fixation and subsequently stained for tight junction proteins. Confocal microscopy was then used to visualise, compare and evaluate staining intensity of the tight junctional complexes in order to determine a standardised workflow of reproducible staining. Results: Positive staining was observed following methanol fixation for claudin-1 and ZO-1 tight junction proteins but no staining was detected for occludin in 16HBE14o- cells. Combinatorial fixation with methanol and acetone also produced consistent positive staining for both occludin and ZO-1 tight junction proteins in these cells. When assessed using primary cells cultured at air-liquid interface, similar positive staining for claudin-1 and ZO-1 was observed following methanol fixation, while similar positive staining for occludin and ZO-1 was observed following the same combinatorial fixation with methanol and acetone. Conclusions: The present study demonstrates the importance of a personalised approach to optimise staining for the visualisation of different tight junction proteins. Of significance, the workflow, once optimised, can readily be translated into primary airway epithelial cell air-liquid interface cultures where it can be used to assess barrier integrity in chronic lung diseases.",
keywords = "Air liquid interface, Airway epithelial cells, Confocal microscopy, Fixation, Tight junctions",
author = "Buckley, {Alysia G.} and Kevin Looi and Thomas Iosifidis and Ling, {Kak Ming} and Sutanto, {Erika N.} and Martinovich, {Kelly M.} and Elizabeth Kicic-Starcevich and Garratt, {Luke W.} and Shaw, {Nicole C.} and Lannigan, {Francis J.} and Larcombe, {Alexander N.} and Graeme Zosky and Knight, {Darryl A.} and Rigby, {Paul J.} and Anthony Kicic and Stick, {Stephen M.}",
year = "2018",
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doi = "10.1186/s12575-018-0070-0",
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Buckley, AG, Looi, K, Iosifidis, T, Ling, KM, Sutanto, EN, Martinovich, KM, Kicic-Starcevich, E, Garratt, LW, Shaw, NC, Lannigan, FJ, Larcombe, AN, Zosky, G, Knight, DA, Rigby, PJ, Kicic, A & Stick, SM 2018, 'Visualisation of Multiple Tight Junctional Complexes in Human Airway Epithelial Cells' Biological Procedures Online, vol. 20, no. 1, 3. https://doi.org/10.1186/s12575-018-0070-0

Visualisation of Multiple Tight Junctional Complexes in Human Airway Epithelial Cells. / Buckley, Alysia G.; Looi, Kevin; Iosifidis, Thomas; Ling, Kak Ming; Sutanto, Erika N.; Martinovich, Kelly M.; Kicic-Starcevich, Elizabeth; Garratt, Luke W.; Shaw, Nicole C.; Lannigan, Francis J.; Larcombe, Alexander N.; Zosky, Graeme; Knight, Darryl A.; Rigby, Paul J.; Kicic, Anthony; Stick, Stephen M.

In: Biological Procedures Online, Vol. 20, No. 1, 3, 01.02.2018.

Research output: Contribution to journalArticle

TY - JOUR

T1 - Visualisation of Multiple Tight Junctional Complexes in Human Airway Epithelial Cells

AU - Buckley, Alysia G.

AU - Looi, Kevin

AU - Iosifidis, Thomas

AU - Ling, Kak Ming

AU - Sutanto, Erika N.

AU - Martinovich, Kelly M.

AU - Kicic-Starcevich, Elizabeth

AU - Garratt, Luke W.

AU - Shaw, Nicole C.

AU - Lannigan, Francis J.

AU - Larcombe, Alexander N.

AU - Zosky, Graeme

AU - Knight, Darryl A.

AU - Rigby, Paul J.

AU - Kicic, Anthony

AU - Stick, Stephen M.

PY - 2018/2/1

Y1 - 2018/2/1

N2 - Background: Apically located tight junctions in airway epithelium perform a fundamental role in controlling macromolecule migration through paracellular spaces. Alterations in their expression may lead to disruptions in barrier integrity, which subsequently facilitates entry of potential bacterial and other pathogens into the host. Furthermore, there is emerging evidence that the barrier integrity of the airway in certain airway inflammatory diseases may be altered. However, there is little consensus on the way this is assessed and measured and the type of cells used to achieve this. Methods: Here, we assessed four fixation methods including; (i) 4% (v/v) paraformaldehyde; (ii) 100% methanol; (iii) acetone or; (iv) 1:1 methanol: acetone. Pre-extraction with Triton X-100 was also performed and assessed on cells prior to fixation with either methanol or paraformaldehyde. Cells were also permeabilized with 0.1% (v/v) Saponin in 1× TBS following fixation and subsequently stained for tight junction proteins. Confocal microscopy was then used to visualise, compare and evaluate staining intensity of the tight junctional complexes in order to determine a standardised workflow of reproducible staining. Results: Positive staining was observed following methanol fixation for claudin-1 and ZO-1 tight junction proteins but no staining was detected for occludin in 16HBE14o- cells. Combinatorial fixation with methanol and acetone also produced consistent positive staining for both occludin and ZO-1 tight junction proteins in these cells. When assessed using primary cells cultured at air-liquid interface, similar positive staining for claudin-1 and ZO-1 was observed following methanol fixation, while similar positive staining for occludin and ZO-1 was observed following the same combinatorial fixation with methanol and acetone. Conclusions: The present study demonstrates the importance of a personalised approach to optimise staining for the visualisation of different tight junction proteins. Of significance, the workflow, once optimised, can readily be translated into primary airway epithelial cell air-liquid interface cultures where it can be used to assess barrier integrity in chronic lung diseases.

AB - Background: Apically located tight junctions in airway epithelium perform a fundamental role in controlling macromolecule migration through paracellular spaces. Alterations in their expression may lead to disruptions in barrier integrity, which subsequently facilitates entry of potential bacterial and other pathogens into the host. Furthermore, there is emerging evidence that the barrier integrity of the airway in certain airway inflammatory diseases may be altered. However, there is little consensus on the way this is assessed and measured and the type of cells used to achieve this. Methods: Here, we assessed four fixation methods including; (i) 4% (v/v) paraformaldehyde; (ii) 100% methanol; (iii) acetone or; (iv) 1:1 methanol: acetone. Pre-extraction with Triton X-100 was also performed and assessed on cells prior to fixation with either methanol or paraformaldehyde. Cells were also permeabilized with 0.1% (v/v) Saponin in 1× TBS following fixation and subsequently stained for tight junction proteins. Confocal microscopy was then used to visualise, compare and evaluate staining intensity of the tight junctional complexes in order to determine a standardised workflow of reproducible staining. Results: Positive staining was observed following methanol fixation for claudin-1 and ZO-1 tight junction proteins but no staining was detected for occludin in 16HBE14o- cells. Combinatorial fixation with methanol and acetone also produced consistent positive staining for both occludin and ZO-1 tight junction proteins in these cells. When assessed using primary cells cultured at air-liquid interface, similar positive staining for claudin-1 and ZO-1 was observed following methanol fixation, while similar positive staining for occludin and ZO-1 was observed following the same combinatorial fixation with methanol and acetone. Conclusions: The present study demonstrates the importance of a personalised approach to optimise staining for the visualisation of different tight junction proteins. Of significance, the workflow, once optimised, can readily be translated into primary airway epithelial cell air-liquid interface cultures where it can be used to assess barrier integrity in chronic lung diseases.

KW - Air liquid interface

KW - Airway epithelial cells

KW - Confocal microscopy

KW - Fixation

KW - Tight junctions

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U2 - 10.1186/s12575-018-0070-0

DO - 10.1186/s12575-018-0070-0

M3 - Article

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JO - Biological Procedures Online

JF - Biological Procedures Online

SN - 1480-9222

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