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Am J Respir Cell Mol Biol. 2020 Mar 24. doi: 10.1165/rcmb.2019-0453MA. [Epub ahead of print]

Single Cell Flow Cytometry Profiling of Bronchoalveolar Lavage in Children.

Author information

1
Royal Children's Hospital, Respiratory and Sleep Medicine, Parkville, Victoria, Australia.
2
Murdoch Children's Research Institute, Respiratory Diseases, Parkville, Victoria, Australia.
3
The University of Melbourne, 2281, Paediatrics, Melbourne, Victoria, Australia; shivanthan.shanthikumar@rch.org.au.
4
Murdoch Childrens Research Institute, 34361, Flow Cytometry, Parkville, Victoria, Australia.
5
Murdoch Childrens Research Institute, Epigenetics, Melbourne, Victoria, Australia.
6
The University of Melbourne, 2281, Paediatrics, Melbourne, Victoria, Australia.
7
Royal Children's Hospital, Respiratory and Sleep Medicine, Melbourne, Australia.
8
Murdoch Children's Research Institute, Parkville, Victoria, Australia.

Abstract

Childhood pulmonary diseases not only cause childhood morbidity and mortality, but can also cause long term pulmonary impairment. The clinical management of many childhood pulmonary diseases is hampered by a limited understanding of the underlying pathophysiological mechanisms. Flow cytometry, which can be used to phenotype individual cell populations or isolate cells for downstream analysis, represents a crucial technology which can help to elucidate the pathophysiology of these conditions. Here, we describe a flow cytometry-based method for purification and characterisation of cell populations in bronchoalveolar lavage (BAL) from children. This includes assessment of the effect of cryopreservation on cell phenotype and frequency, a knowledge gap recently identified by an American Thoracic Society report on flow cytometry in lung samples. To our knowledge, this is the first study to simultaneously quantify alveolar macrophages, T cells (CD4 and CD8), B cells, NK cells, dendritic cells, granulocytes and monocytes (CD16+/CD16-) in the BAL of children. The protocols described can be used to advance investigation of the pathophysiology of childhood pulmonary diseases.

KEYWORDS:

Bronchoalveolar Lavage; Cryopreservation; Flow Cytometry; Pediatrics; cystic fibrosis

PMID:
32207982
DOI:
10.1165/rcmb.2019-0453MA

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