Cytexpert version 2

Cells were seeded in 6-well plates, treated and incubated with 10 µM EdU for 1 h at the end of treatment. Cells were harvested by trypsinization and labelled using the Click-iT Plus EdU Alexa Fluor 647 Flow Cytometry Assay Kit (Invitrogen) according to the manufacturer’s instructions. For the analysis of DSB levels, cells were then stained protected from light with γH2AX primary antibody (Cell Signaling, 9718S, 1:200) in 1% BSA in PBS for 1 h at room temperature, washed once and incubated for 30 min with Alexa Fluor 488-conjugated goat anti-rabbit secondary antibody (Molecular Probes/Thermo Fisher, A11034, 1:500) in 1% BSA in PBS. For DAPI staining, cell pellets were resuspended in 1 μg/mL DAPI solution in PBS and incubated for 10 min. Cells were washed in PBS and analysed immediately after staining on Cytoflex LX (Beckman Coulter), using CytExpert version 2.3 (Beckman Coulter) for data collection. Post-acquisition analysis was performed in FlowJo software version 10 (BD Biosciences).

FACS analysis experiments for determination of BMPR2 expressing cells were performed on a CytoFlex Flow Cytometer (Beckman Coulter). Cells were fixed with 1% PFA before blocking with Human TruStain FcX™ (BioLegend, San Diego, CA, USA) and subsequently labeled with eBioscience™ Fixable Viability Dye eFluor™ 450 (Invitrogen, Waltham, MA, USA) to select for viable cells. Subsequently the cells were stained with BMPR2 antibody (#ab78422, Abcam, Cambridge, UK) and PE goat anti mouse IgG as secondary antibody (#405307 BioLegend) according to the manufacturer’s recommendation. Raw CytoFLEX data were processed using CytExpert version 2.3 (Beckman Coulter).

We isolated the immune cells from the spleen of mice using a cell strainer and the Percoll gradient centrifugation method as previously described. For the incubation of different antibodies for cell-sorting of different immune cells, three panels were used in the experiment: panel 1 (CD45, CD3, and CD4), panel 2 (CD45, CD64, CD11b, and Ly6G), and panel 3 (CD45, CD4, CD3, and FOXP3). For the arrangement of the channels for cell sorting of the cells in each tube, the PC-A750, APC, PC5.5, and FITC channels were used for cell sorting in tube 1; the APC-A750, PC5.5, APC, and PE channels were used for cell sorting in tube 2; and the PC5.5, APC, APC-A750, and PE channels were used for cell sorting in tube 3. A FACS Canto Flow Cytometer (Beckman Coulter, Brea, CA, USA) was used for flow cytometry analysis. The percentage (%) of the total CD45⁺ populations represents the abundances of specific groups of immune cells, such as CD11b⁺Ly6G⁺ neutrophils, CD11b⁺CD64⁺ macrophages, CD4⁺Foxp3⁺ regulatory T cells, CD3⁺CD4⁺ T helper cells, and CD3⁺CD8⁺ cytotoxic T cells. The CytExpert version 2.3 (Beckman Coulter) was used for the analysis of the flow cytometry data.