Cd133 1 pecy7

Cell apoptosis was evaluated using Pacific Blue Annexin V (Biolegend, 640918) and 7-AAD Viability Staining Solution (BioLegend, 420403). In detail, GE-HSPCs were stained with the following antibodies mix for 15 min at 4°C: CD34 PE (Miltenyi Biotec 130-081-00), CD90 APC (BD Biosciences, 559869), and CD133/1 PECy7 (Miltenyi Biotec, 130-101-652). Cells were then washed with diluted 1:10 10X Annexin V Binding Buffer (BD Pharmingen, 556454) upon surface marker staining and stained with Pacific Blue Annexin V (Biolegend, 640918) and 7-AAD Viability Staining Solution (BioLegend, 420403) for 15 min at RT in the dark. After staining, cells were washed in 1X Annexin V Binding Buffer and acquired in 10 min. All samples were run on BD FACSCanto II cytometer (BD Biosciences). At least 10,000 were recorded. Analysis of flow cytometry results was performed using FlowJo software.

0.5–1 × 10⁵ cells were treated with 2 μM EdU for 4 h in culture (Thermo Fisher Scientific, C10636), washed with PBS + 1% BSA, and stained with the following antibodies mix for 15 min at 4°C: CD34 PE (Miltenyi Biotec 130-081-00), CD90 APC (BD Biosciences, 559869), and CD133/1 PECy7 (Miltenyi Biotec, 130-101-652). Cells were then fixed with 100 μL of Click-iT fixative for 15 min at RT upon cell-surface staining to distinguish the different HSPC subsets. After washing with PBS + 1% BSA, cells were permeabilized with 100 μL 1X Click-iT saponin for 15 min. Detection of EdU-DNA was performed by incubating cells with 500 μL Click-iT Plus reaction cocktail for 30 min at RT protected from light. Cells were subsequently washed with PBS + 1% BSA before overnight DNA staining with Hoechst at RT protected from light. Fluorescence was measured by flow cytometry on FACSymphony A5 SORP (BD Biosciences). Analysis of flow cytometry results was performed using FlowJo software.