Fc500 flow cytometer

For apoptosis and cell cycle analysis, 2 × 10⁵ MLE-12 cells and MenSCs were seeded into the lower and upper transwell chambers of 6-well plates, respectively. An Annexin V/PI detection kit (BD Biosciences, CA, USA) was used to detect MLE-12 apoptosis, and a Cell Cycle and Apoptosis analysis kit (Beyotime Biotechnology, Haimen, China) was used to detect the MLE-12 cell cycle. Both experiments were performed according to the manufacturer’s instructions using a FC500 flow cytometer, and the data were analyzed using FlowJo software (Tree Star, OR, USA).
To determine the number of lung epithelial cells in lung tissues, flow cytometry was performed. The lung tissues were cut into small pieces and digested with type 1 collagenase for 1 h at 37 °C. Then a single-cell suspension was obtained by grinding the digested tissue through a 100-μm filter (BD Biosciences, CA, USA) before being centrifuged at 300×g for 10 min at 4 °C, and red blood cells were lysed twice. Subsequently, the cells were resuspended with stain buffer (BD Biosciences) and incubated with antibodies listed in Additional file 5, Table S1 for 30 min at 4 °C. Subsequently, the cells were washed twice with stain buffer before being analyzed with a multicolor flow cytometer (BD Biosciences). The data were analyzed using FlowJo software.

EBs were pelleted by centrifugation and washed with PBS. EBs were disaggregated by the addition of 1ml Accumax cell dissociation buffer (Millipore, Billerica, MA) and incubated at 37°C for 30 minutes. Single-cell suspensions were collected and washed with PBS and incubated with the indicated antibodies. CD41-PE, FLK1-PE, ckit-APC, PDGFRα-biotin, and avidin-APC-Cy7 were obtained from Biolegend (San Diego, CA). Stained cells were subsequently assessed using Beckman Coulter FC500 Flow Cytometer (Brea, CA) and data was analyzed using Flowjo software (Tree Star, Ashland, OR).

Cells were co-labeled with propidium iodide, to gate on viable populations. During data analysis, MSCs were excluded from MDMs by CD90 positive cell exclusion, and from Th cells by CD4 negative cell exclusion. Cells were characterized using a FC500 flow cytometer and analyzed on FlowJo (Treestar). Antibodies used were: FITC-CD4 (OKT4), PE-Cy7-CD14 (61D3), PE-CD206 (19.2), PE-CD127 (eBioRDR5) and PE-Cy7-CD25 (BC96) (eBioscience); FITC-CD163 (GHI/61) and APC-CD90 (5E10) (Biolegend, San Diego, CA).