Senescence associated beta galactosidase sa β gal staining kit

StemSpan SFEM II, Serum-free Medium for Expansion of Hematopoietic Cells was sourced from STEMCELL Technologies (Seattle, WA, USA). The lymphocyte separation medium was sourced from Axis-Shield Diagnostics Ltd. (US). The red blood cell lysis buffer, BCA protein assay kit, senescence-associated beta-galactosidase (SA-β-gal) staining kit, cell lysis buffer, lipid peroxidation (MDA) assay kit, superoxide dismutase (SOD) assay kit, and reactive oxygen species (ROS) assay kit were sourced from Beyotime Biotech Co., Ltd. (China), while the EdU kit was sourced from RiboBio Co., Ltd. (China). The reverse transcription kit was sourced from TOYOBO biotech Co. (Japan) and the H4434 medium and complete hematopoietic cell culture medium were sourced from STEMCELL Technologies (US). The anti-P16, anti-P19, anti-P21 and anti-P53 monoclonal antibodies were sourced from Cell Signaling Technology (US).

EPC senescence was determined using a senescence-associated beta-galactosidase (SA-β-gal) staining kit (Beyotime, Haimen, China). EPCs were fixed with a fixation buffer at room temperature for 15 min. After washing with PBS 3 times, the cells were incubated with a working solution at 37 °C without CO₂ for 12 h. PBS was added to stop the reaction. The ratio of the β-gal-positive cells to the total number of cells was determined under light microscopy.