Populations of primary MEFs (2 x 105 (link) per 10 cm dish) were counted by trypan blue exclusion every third day. SA-β-Gal staining was performed according to the manufactures instructions (Cell Signaling, SA-β-Gal staining kit (#9860)) and images were acquired with a stereomicroscope (Leica MZ 16 1FA). DAPI staining was used to identify nuclei.
For FACS-based determination of senescence, lysosomal alkalinization was induced by treating cells with 100 nM bafilomycin A1 for 1 h in fresh cell culture medium (2 ml per 35 mm dish) at 37 °C. 33 μl of 2 mM C12FDG (Thermofisher, ref. D2893) working solution was added to the cell culture medium to obtain a final concentration of 33 μM. After 1h of incubation, cells were washed, trypsinised and collected on a Gallios flow cytometer (Beckman Coulter) using Kaluza Acquisition software. Data were analysed with FlowJo software.
For FACS-based determination of senescence, lysosomal alkalinization was induced by treating cells with 100 nM bafilomycin A1 for 1 h in fresh cell culture medium (2 ml per 35 mm dish) at 37 °C. 33 μl of 2 mM C12FDG (Thermofisher, ref. D2893) working solution was added to the cell culture medium to obtain a final concentration of 33 μM. After 1h of incubation, cells were washed, trypsinised and collected on a Gallios flow cytometer (Beckman Coulter) using Kaluza Acquisition software. Data were analysed with FlowJo software.