Cargando…

Quantifying Intracellular Viral Pathogen: Specimen Preparation, Visualization and Quantification of Multiple Immunofluorescent Signals in Fixed Human Airway Epithelium Cultured at Air-Liquid Interface

Infection control and aggressive antibiotic therapy play an important role in the management of airway infections in individuals with cystic fibrosis (CF). The responses of airway epithelial cells to pathogens are likely to contribute to the pathobiology of CF lung disease. Primary airway epithelial...

Descripción completa

Detalles Bibliográficos
Autores principales: Wong, Sharon L., Pandzic, Elvis, Kardia, Egi, Allan, Katelin M., Whan, Renee M., Waters, Shafagh A.
Formato: Online Artículo Texto
Lenguaje:English
Publicado: MDPI 2022
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9605096/
https://www.ncbi.nlm.nih.gov/pubmed/36294807
http://dx.doi.org/10.3390/jpm12101668
_version_ 1784817980691972096
author Wong, Sharon L.
Pandzic, Elvis
Kardia, Egi
Allan, Katelin M.
Whan, Renee M.
Waters, Shafagh A.
author_facet Wong, Sharon L.
Pandzic, Elvis
Kardia, Egi
Allan, Katelin M.
Whan, Renee M.
Waters, Shafagh A.
author_sort Wong, Sharon L.
collection PubMed
description Infection control and aggressive antibiotic therapy play an important role in the management of airway infections in individuals with cystic fibrosis (CF). The responses of airway epithelial cells to pathogens are likely to contribute to the pathobiology of CF lung disease. Primary airway epithelial cells obtained from individuals with CF, cultured and differentiated at air-liquid interface (ALI), effectively mimic the structure and function of the in vivo airway epithelium. With the recent respiratory viral pandemics, ALI cultures were extensively used to model respiratory infections in vitro to facilitate physiologically relevant respiratory research. Immunofluorescence staining and imaging were used as an effective tool to provide a fundamental understanding of host–pathogen interactions and for exploring the therapeutic potential of novel or repurposed drugs. Therefore, we described an optimized quantitative fluorescence microscopy assay for the wholemount staining and imaging of epithelial cell markers to identify distinct cell populations and pathogen-specific targets in ALI cultures of human airway epithelial cells grown on permeable support insert membranes. We present a detailed methodology using a graphical user interface (GUI) package to quantify the detected signals on a tiled whole membrane. Our method provided an imaging strategy of the entire membrane, overcoming the common issue of undersampling and enabling unbiased quantitative analysis.
format Online
Article
Text
id pubmed-9605096
institution National Center for Biotechnology Information
language English
publishDate 2022
publisher MDPI
record_format MEDLINE/PubMed
spelling pubmed-96050962022-10-27 Quantifying Intracellular Viral Pathogen: Specimen Preparation, Visualization and Quantification of Multiple Immunofluorescent Signals in Fixed Human Airway Epithelium Cultured at Air-Liquid Interface Wong, Sharon L. Pandzic, Elvis Kardia, Egi Allan, Katelin M. Whan, Renee M. Waters, Shafagh A. J Pers Med Protocol Infection control and aggressive antibiotic therapy play an important role in the management of airway infections in individuals with cystic fibrosis (CF). The responses of airway epithelial cells to pathogens are likely to contribute to the pathobiology of CF lung disease. Primary airway epithelial cells obtained from individuals with CF, cultured and differentiated at air-liquid interface (ALI), effectively mimic the structure and function of the in vivo airway epithelium. With the recent respiratory viral pandemics, ALI cultures were extensively used to model respiratory infections in vitro to facilitate physiologically relevant respiratory research. Immunofluorescence staining and imaging were used as an effective tool to provide a fundamental understanding of host–pathogen interactions and for exploring the therapeutic potential of novel or repurposed drugs. Therefore, we described an optimized quantitative fluorescence microscopy assay for the wholemount staining and imaging of epithelial cell markers to identify distinct cell populations and pathogen-specific targets in ALI cultures of human airway epithelial cells grown on permeable support insert membranes. We present a detailed methodology using a graphical user interface (GUI) package to quantify the detected signals on a tiled whole membrane. Our method provided an imaging strategy of the entire membrane, overcoming the common issue of undersampling and enabling unbiased quantitative analysis. MDPI 2022-10-07 /pmc/articles/PMC9605096/ /pubmed/36294807 http://dx.doi.org/10.3390/jpm12101668 Text en © 2022 by the authors. https://creativecommons.org/licenses/by/4.0/Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https://creativecommons.org/licenses/by/4.0/).
spellingShingle Protocol
Wong, Sharon L.
Pandzic, Elvis
Kardia, Egi
Allan, Katelin M.
Whan, Renee M.
Waters, Shafagh A.
Quantifying Intracellular Viral Pathogen: Specimen Preparation, Visualization and Quantification of Multiple Immunofluorescent Signals in Fixed Human Airway Epithelium Cultured at Air-Liquid Interface
title Quantifying Intracellular Viral Pathogen: Specimen Preparation, Visualization and Quantification of Multiple Immunofluorescent Signals in Fixed Human Airway Epithelium Cultured at Air-Liquid Interface
title_full Quantifying Intracellular Viral Pathogen: Specimen Preparation, Visualization and Quantification of Multiple Immunofluorescent Signals in Fixed Human Airway Epithelium Cultured at Air-Liquid Interface
title_fullStr Quantifying Intracellular Viral Pathogen: Specimen Preparation, Visualization and Quantification of Multiple Immunofluorescent Signals in Fixed Human Airway Epithelium Cultured at Air-Liquid Interface
title_full_unstemmed Quantifying Intracellular Viral Pathogen: Specimen Preparation, Visualization and Quantification of Multiple Immunofluorescent Signals in Fixed Human Airway Epithelium Cultured at Air-Liquid Interface
title_short Quantifying Intracellular Viral Pathogen: Specimen Preparation, Visualization and Quantification of Multiple Immunofluorescent Signals in Fixed Human Airway Epithelium Cultured at Air-Liquid Interface
title_sort quantifying intracellular viral pathogen: specimen preparation, visualization and quantification of multiple immunofluorescent signals in fixed human airway epithelium cultured at air-liquid interface
topic Protocol
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9605096/
https://www.ncbi.nlm.nih.gov/pubmed/36294807
http://dx.doi.org/10.3390/jpm12101668
work_keys_str_mv AT wongsharonl quantifyingintracellularviralpathogenspecimenpreparationvisualizationandquantificationofmultipleimmunofluorescentsignalsinfixedhumanairwayepitheliumculturedatairliquidinterface
AT pandzicelvis quantifyingintracellularviralpathogenspecimenpreparationvisualizationandquantificationofmultipleimmunofluorescentsignalsinfixedhumanairwayepitheliumculturedatairliquidinterface
AT kardiaegi quantifyingintracellularviralpathogenspecimenpreparationvisualizationandquantificationofmultipleimmunofluorescentsignalsinfixedhumanairwayepitheliumculturedatairliquidinterface
AT allankatelinm quantifyingintracellularviralpathogenspecimenpreparationvisualizationandquantificationofmultipleimmunofluorescentsignalsinfixedhumanairwayepitheliumculturedatairliquidinterface
AT whanreneem quantifyingintracellularviralpathogenspecimenpreparationvisualizationandquantificationofmultipleimmunofluorescentsignalsinfixedhumanairwayepitheliumculturedatairliquidinterface
AT watersshafagha quantifyingintracellularviralpathogenspecimenpreparationvisualizationandquantificationofmultipleimmunofluorescentsignalsinfixedhumanairwayepitheliumculturedatairliquidinterface