Additional file 1 of Neutrophil extracellular traps are present in the airways of ENaC-overexpressing mice with cystic fibrosis-like lung disease
Creators: name: Tucker, Samantha L.
name: Demba Sarr
name: Rada, Balázs
Publisher: name: figshare
Year: 2021
Subjects: subject: Medicine
subject: Microbiology
subject: FOS: Biological sciences
subject: FOS: Biological sciences
subject: Physiology
subject: 39999 Chemical Sciences not elsewhere classified
subject: FOS: Chemical sciences
subject: FOS: Chemical sciences
subject: Immunology
subject: FOS: Clinical medicine
subject: FOS: Clinical medicine
subject: Cancer
subject: 110309 Infectious Diseases
subject: FOS: Health sciences
subject: FOS: Health sciences

description: Additional file 1: Figure S1. Genotyping of β-ENaC-Tg mice by PCR. Mice were identified as either WT C57BL/6 or heterozygous for over-expression of β-ENaC using PCR of the intron region of Scnn1b. (A) A schematic representation of the expected PCR product sizes for both WT and transgenic β-ENaC-Tg. (B) A representative gel electrophoresis comparing the final PCR product of WT and heterozygous β-ENaC-Tg mice. Figure S2. Flow cytometry gating strategy: myeloid cells. A representative schematic depicting the method for selecting myeloid cell populations present within the BAL of uninfected WT and β-ENaC-Tg mice. Single cells, negative for zombie aqua fixable viability dye were considered live. The myeloid cell marker CD11b was used to separate myeloid cells from other cell types. The CD11b+ cells were considered the parent cell population for all cell-types measured. The markers used for each cell type is as follows: Neutrophils (CD11b+, CD115−, Ly6G+), Eosinophils (CD11b+, CD115−, Ly6G+); Monocytes (CD11b+, CD115+, Ly6G+); Inflammatory Monocytes (CD11b+, CD115+, Ly6GHigh), Macrophages (CD11b+, F4/80+): Dendritic Cells (CD11b+,CD11c+,F4/80−); Alveolar Macrophages (CD11b+, F4/80−, CD115+, CD11c−); Inflammatory Macrophages (CD11b+/F4/80+, CD115+, CD11c+). Figure S3. Results of the multiplex cytokine Bioplex array. Multiplex bead-based ELISA was used to measure the concentration of 23 cytokines and chemokines in the BAL supernatant of β-ENaC-Tg mice and their WT littermate controls at 6 and 8 weeks old (n = 9). The results show significant increases in neutrophil-associated chemokines including KC, MIP-1α, MIP-1β, and G-CSF for the β-ENaC-Tg mice at either 6 weeks, 8 weeks, or both. Figure S4. Flow cytometry gating strategy: citrullinated histone. A representative schematic depicting the method for selecting neutrophils undergoing histone citrullination in the BAL of uninfected WT and β-ENaC-Tg mice. Single cells, negative for zombie aqua fixable viability dye were considered live. The myeloid cell marker CD11b was used to separate myeloid cells from other cell types. Neutrophils positive for citrullination were CD11b+, CD115−, Ly6G+, histone H3 (citrulline R2+R8+R17)+. Figure S5. Analysis of citrullination positive cell populations in the BAL of 6 and 8 week old mice. Flow cytometry of BAL cells showed significantly more neutrophils (Ly6G+, CD11b+) cells and more total citrullination (CitH3) positive cells present in the β-ENaC-Tg mice compared to WT littermate controls. There was not a significant difference between percent of citrullination positive neutrophils in the BAL (CitH3+, Ly6G+).