Apoptosis of HSCs was detected by flow cytometry analysis using eBioscience™ Annexin V Apoptosis Detection Kit (Thermo Fisher Scientific, CA, USA) according to the manufacturer’s protocol. After being treated with extracellular vesicles or control solution for 24 h, the cells were collected and washed three times in pre-cold PBS. The samples were subsequently stained with fluorochrome-conjugated Annexin V for 15 min at room temperature, followed by washing with Binding Buffer. Finally, cells were treated with propidium iodide staining solution and detected in Accuri C6 flow cytometry (BD Biosciences, MD, USA).
Cell cycle analysis was performed using FxCycle™ PI/RNase Staining kit (Thermo Fisher Scientific, CA, USA) according to the manufacturer’s instructions. HSCs treated with exosome or control solution were washed twice with ice-cold PBS and resuspended in pre-cold 70% ethanol. After being washed with PBS and treated with DNase-free Rnase A for 30 min in 37 °C, the samples were incubated for 30 min at room temperature. Results were analyzed by the Modfit software (Verity Software House, CA, USA).
Cell cycle analysis was performed using FxCycle™ PI/RNase Staining kit (Thermo Fisher Scientific, CA, USA) according to the manufacturer’s instructions. HSCs treated with exosome or control solution were washed twice with ice-cold PBS and resuspended in pre-cold 70% ethanol. After being washed with PBS and treated with DNase-free Rnase A for 30 min in 37 °C, the samples were incubated for 30 min at room temperature. Results were analyzed by the Modfit software (Verity Software House, CA, USA).