Annexin 5 buffer

To differentiate live cells from dead and apoptotic cells, the hGL5 cells were stained with 1:10,000 BD Horizon Fixable Viability Stain 660 (BD Biosciences, San Jose, CA, USA) and 5 μL Annexin V-phycoerythrin antibody per tube (BioLegend, San Diego, CA, USA) for 15 min in a modified staining buffer containing culture medium and Annexin V buffer (BioLegend) at a 1:1 ratio on ice. The labeled cells were washed with modified staining buffer and analyzed using flow cytometry on a BD LSRFortessa Cell Analyzer (BD Biosciences). Before flow cytometry, 50 μL of CountBright Absolute counting beads (Invitrogen) were mixed with the cell samples in a calibration tube, according to the manufacturer’s instructions (target cell count in a tube = number of target cells/number of counted beads in each sample × assigned bead count in the 50 μL of counting beads) [36 (link)].

EndoC-βH1 cells were trypsinized and washed three times in PBS. Cell death was assessed by incubating the cells with annexin-V antibody (APC, #350520, Biolegend) for 15 min at room temperature in the dark in annexin-V buffer (#422201, Biolegend). Propidium iodide was added before fluorescence-activated cell sorting (FACS) analysis. For proliferation studies, cells were fixed and permeabilized with a transcription factor staining buffer set (#00–5523–00, Thermo Fisher Scientific) according to the manufacturer’s instructions. Cells were then incubated with Ki67 antibody (PerCP/Cy5.5, #B238642, Biolegend) for 30 min at room temperature in the dark in permeabilization buffer and then with DAPI (BD Biosciences) for 10 min at room temperature in the dark. FACS analysis was carried out using a FACS Aria III (BD Biosciences). Data were analyzed using FlowJo 10.7 software (RRID:SCR_008520).

The AEC pellet was resuspended in 10 μl of FC block solution, and after 15 min, Annexin V antibody (Alexa Fluor 647, BioLegend 1:20) diluted in 100 μl Annexin V buffer was added (BioLegend, Cat No 640922). The samples were incubated for 15–25 min at 4 °C. All stained cell suspensions were filtered in 5 ml polystyrene tubes and analyzed by a BD LSRFortessa^TM flow cytometer to quantify the proportion of apoptotic AEC.