Flow cytometry analysis of lung immune cell populations

For flow cytometry experiments, pups were culled on day 7 (n = 6) and whole lungs were minced mechanically before digestion with collagenase D (1 mg/ml) at 37 °C for 20 minutes. Lung homogenate was passed through 70-μm and 40-μm filters to obtain a single cell suspension prior to staining. After blocking Fc receptors, cells were stained using a combination of surface markers and intracellular stains. These included CD45, CD25, NKp46, Foxp3, MHCII and F4/80 purchased from eBioscience, CD4, CD3, CD8, B220 and CD11c from Biolegend, and CD11b, CD103, Ly6C, Ly6G and SiglecF from BD. A full list of antibodies is presented in Table 1. Natural killer cells were identified as the CD45⁺NKp46⁺CD3^– population, neutrophils were identified as the CD45⁺Ly6G^hiCD11b^hiCD11C^hiF4/80^– population, CD11bc dendritic cells were identified as the CD45⁺CD103^loCD11C^hiLy6C^–F4/80^intMHCII^hiCD11b^hiSiglec-F^– population, CD103c dendritic cells were identified as the CD45⁺CD103^hiCD11C^hiLy6C^–F4/80^loMHCII^hiCD11b^loSiglec-F^– population and interstitial macrophages (imacs) were identified as the CD45⁺CD103^loCD11C^loLy6C^loF4/80^intMHCII^hiCD11b^loSiglec-F^– population. The representative FACS plots for these populations are shown in Fig. 3a, d, f. Analysis was performed using a BD FACS Canto II Flow (BD Biosciences, NSW, Australia).

Antibody information for fluorescence-activated cell sorting analysis

Hart’s staining on PND14 (n = 6–8). a Representative images for Hart’s staining. Scale bar = 100 μm. b, c Secondary septal crest density was decreased in the saline-treated injured group. Early intravenous (b) and intratracheal (c) injection of hAECs restored the tissue-to-air space ratio. d Both early and late treatment restored septal crest density. Data expressed as mean ± standard error of mean (SEM). Statistical significance determined with one-way ANOVA accompanied by the Bonferroni post-hoc test. *p < 0.05, **p < 0.01, ****p < 0.0001. hAEC human amnion epithelial cell, IT intratracheal, IV intravenous