Characterizing Immune Cell Populations in Lung Tissue

Inflammatory cells in lung tissue were analyzed to identify eosinophils (CD45+CD11b+Ly6C-SiglecF+), Th17 cells (CD45+CD4+IL-17A+), regulatory T (Treg) cells (CD45+CD4+CD25+Foxp3+) by flow cytometry as previously described (Zhao et al., 2013 (link); Cossarizza et al., 2017 (link); Dong et al., 2018 (link)). Briefly, the excised right lung was digested by using lung dissociation kits (MiltenyiBiotec Technology & Trading Co. Ltd, Shanghai, China) to prepare single cell suspensions following the instructions. And then the cells were stained with the monoclonal anti-murine ﬂuorochrome-conjugated Abs and detected by an Attune NxT instrument (Life Technology). Besides, the antibodies used were bought from BioLegend, including eflour506-labeled anti-CD45, Zombie NIR™ Fixable Viability Kit, Brilliant Violet 421™-labeled anti-CD11b, APC-labeled anti-Siglec-F, PE-labeled anti-Ly-6C, APC-labeled anti-CD25, FITC-labeled anti-CD4, PE-labeled anti-IL-17. PE-labeled anti-Foxp3 was purchased from eBioscience.

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Yi L., Cui J., Wang W., Tang W., Teng F., Zhu X., Qin J., Wuniqiemu T., Sun J., Wei Y, & Dong J. (2020). Formononetin Attenuates Airway Inﬂammation and Oxidative Stress in Murine Allergic Asthma. Frontiers in Pharmacology, 11, 533841.

Publication 2020

Attune NxT instrument Zombie NIR™ Fixable Viability Kit Brilliant Violet 421™-labeled anti-CD11b FITC-labeled anti-CD4 PE-labeled anti-Foxp3

Antibodies Cd11b Cd25 Cells Eosinophils Fitc Flow cytometry Il 17 Il 17a Inflammatory lung Lung Lung inflammatory Murine Regulatory t cells Siglec Th17 cells Tissue Violet

Corresponding Organization : Fudan University

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Variable analysis

independent variables

Inflammatory cell types in lung tissue (eosinophils, Th17 cells, regulatory T (Treg) cells)

dependent variables

Percentage of eosinophils (CD45+CD11b+Ly6C-SiglecF+)
Percentage of Th17 cells (CD45+CD4+IL-17A+)
Percentage of regulatory T (Treg) cells (CD45+CD4+CD25+Foxp3+)

control variables

Lung tissue sample preparation (digestion using lung dissociation kits)
Staining of cells with monoclonal anti-murine fluorochrome-conjugated antibodies
Detection of cells using Attune NxT instrument

positive controls

Not explicitly mentioned

negative controls

Not explicitly mentioned

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