Hoechst

Cells were incubated with APC/Cy7 anti-mouse Ly-6A/E (Sca-1) (BioLegend, San Diego, CA, USA, 108125, dilution 1:200), FITC anti-mouse CD117 (c-Kit) (BioLegend, 105805; dilution 1:200), AlexaFluor488 anti-mouse PDGFRβ (Becton Dickinson, Heidelberg, Germany 558427, dilution 1:200), and Hoechst 33342 (Sigma Aldrich, Hamburg, Germany; dilution 1:10,000) for 1 h at 4 °C in the dark. For the proliferation analysis, MACS-sorted cells were fixed with chilled ethanol and incubated at 20 °C for 2 h followed by APC anti-mouse Ki67 (BioLegend, 652405, dilution 1:200) and Hoechst for 30 min at room temperature (RT) in the dark. For the apoptosis analysis, MACS-sorted cells were resuspended in annexin V binding buffer (BioLegend, 422201) and stained with Annexin V 647 (BioLegend, 640911, dilution 1:20) and Zombie Green (BioLegend, 423111, dilution 1:1000) for 10 min in the dark. Samples were washed twice with PBS and analyzed by flow cytometry (BD FACS Canto II and BD FACS Aria III).

MSCs were labeled with 1 μM Calcein-AM and 1 μg/mL of Hoechst (BioLegends) and imaged at 10X magnification. Morphologies and areas (cell body and nucleus) were quantified using an ImageJ shape factor parameter plugin. Shape factor of 1 represents a circle, whereas shape factor of 0 represents a line.