β-galactosidase staining

Cells were stained for senescence associated β-Galactosidase activity according to the manufacturer’s protocol (Senescence β-Galactosidase Staining from Cell Signaling #9860). Briefly, growth media was removed and cells were washed once with 1X PBS. 1 mL of 1X Fixative solution was added to each well and allowed to fix for 15 mins at room temperature. Fixed cells were washed twice with 1X PBS. To stain the cells, 1ml of β-Galactosidase Staining Solution was added to each well. Plates were carefully sealed with parafilm to avoid evaporation and incubated at 37°C for 48 hours in a dry incubator devoid of CO₂. Images were taken with Olympus IX73. ImageJ was used to quantify the cells stained for senescence associated β-Galactosidase activity.