Quantification of airway fibrosis in asthma by flow cytometry

Abstract

Airway fibrosis is a prominent feature of asthma, contributing to the detrimental consequences of the disease. Fibrosis in the airway is the result of collagen deposition in the reticular lamina layer of the subepithelial tissue. Myofibroblasts are the leading cell type involved with this collagen deposition. Established methods of collagen deposition quantification present various issues, most importantly their inability to quantify current collagen biosynthesis occurring in airway myofibroblasts. Here, a novel method to quantify myofibroblast collagen expression in asthmatic lungs is described. Single cell suspensions of lungs harvested from C57BL/6 mice in a standard house dust mite model of asthma were employed to establish a flow cytometric method and compare collagen production in asthmatic and non-asthmatic lungs. Cells found to be CD45 αSMA , indicative of myofibroblasts, were gated, and median fluorescence intensity of the anti-collagen-I antibody labeling the cells was calculated. Lung myofibroblasts with no, medium, or high levels of collagen-I expression were distinguished. In asthmatic animals, collagen-I levels were increased in both medium and high expressers, and the number of myofibroblasts with high collagen-I content was elevated. Our findings determined that quantification of collagen-I deposition in myofibroblastic lung cells by flow cytometry is feasible in mouse models of asthma and indicative of increased collagen-I expression by asthmatic myofibroblasts. © 2018 International Society for Advancement of Cytometry.

Best Practices for Preparing a Single Cell Suspension from Solid Tissues for Flow Cytometry

“…However, fixation itself may affect antigenicity by changing protein conformation, so antibody recognition should also be evaluated in pilot experiments. Fixation physically stabilizes cells in their current state while preventing the fragmentation of the newly dead cells . Mild paraformaldehyde fixation also preserves light scatter properties critical to heterogeneous cell populations and helps to prevent cells from sticking together or to the plate or tube in which they are stained.…”

“…Mild paraformaldehyde fixation also preserves light scatter properties critical to heterogeneous cell populations and helps to prevent cells from sticking together or to the plate or tube in which they are stained. Fixation has been shown to be especially crucial for fragile/heterogeneous populations such as lung single cells . Cryopreservable live/dead dye should be applied to cells before fixing to allow for discrimination of these two populations during data analysis.…”

DEK‐targeting aptamer DTA‐64 attenuates bronchial EMT‐mediated airway remodelling by suppressing TGF‐β1/Smad, MAPK and PI3K signalling pathway in asthma

“…To detect the production of α‐SMA in the lung, lung tissues were incubated with 1 mg/mL Collagenase A (#17100017; Gibco, Gaithersburg, MD, USA), 1500 kU/mL DNase I (#D5025‐150KU; Sigma‐Aldrich Chemical Co., St. Louis, MO, USA) and CaCl 2 (0.025M) at 37℃ for 4 hours to prepare single cells suspension, as previously reported 14 . For detecting the cytokines levels in the mediastinal lymph node (mLN) and spleen, lymph nodes and spleen were isolated and grinded with ground glasses, and cells were prepared for further detection by flow cytometry.…”

Using the Autofluorescence Finder on the Sony ID7000TM Spectral Cell Analyzer to Identify and Unmix Multiple Highly Autofluorescent Murine Lung Populations

“…Populations in the non-hematopoietic/non-EC subset were then defined as pericytes (PDGFRb+/CD146+) ( Figure 5S ) ( Hung et al, 2013 ) and epithelial goblet cells (MUC2+/EPCAM+) ( Figure 5T ) ( Tawiah et al, 2018 ). Myofibroblasts expressing low or high amounts of collagen-1 ( Figure 5V ) were identified after selecting for α-SMA + cells ( Figure 5U ) ( Reichard et al, 2018 ). When antigen expression was unclear, fluorescence minus one (FMO) controls were utilized to set gates ( Figure 6 ).…”