Cell event senescence green probe

Cell Event Senescence Green flow cytometry assay kit (Thermofisher Scientific, Waltham MA, USA) was used according to the manufacturer’s instruction. Briefly DCs were first stained for cell surface marker CD11c using regular staining protocol discussed in detail in the following sections. After the last wash, cells were fixed with 4% paraformaldehyde (PFA) for 10 minutes at room temperature followed by washing to remove the fixative solution. Cells were then incubated with Cell Event Senescence Green probe (Thermofisher Scientific, Waltham MA, USA) at 1:500 dilution for 2 hours at 37°C with no CO2. Cells were then washed with PBS, 2% FBS and data acquired by MACSQuant analyzer machine and MACSQuantify software (Miltenyi Biotech Auburn, CA, USA).

After the treatment (24 and 48 h), the spheroids were washed with PBS and fixated with a 2% paraformaldehyde solution for 10 min. The spheroids were washed in a 10% BSA solution in order to remove the fixation solution and then proceeded to stain the spheroids with the CellEvent™ Senescence Green Probe provided by the CellEvent™ Senescence Green Detection Kit (ThermoFisher Scientific, Waltham, MA USA) and were incubated for 2-and-a-half hours at 37 °C without CO₂ and in the absence of light. After incubation, the spheroids were washed with PBS and the fluorescence was measured using λexc = 485 nm and λem = 535 nm with the Mithras LB 940 plate reader.

Cells were isolated from gingival tissues by enzymatic digestion as described above. Then single cell suspension was used to detect SA-β-gal in DCs by flow cytometry. Cell Event Senescence Green flow cytometry assay kit (Thermofisher Scientific, Waltham MA, USA) was used according to the manufacturer’s instruction. Briefly DCs were first stained for cell surface marker, anti-mouse CD11c APC; clone N418 (Invitrogen; Cat#17-0114-82) using regular staining protocol discussed in detail in the above section. After the last wash, cells were fixed with 4% paraformaldehyde (PFA) for 10 minutes at room temperature followed by washing to remove the fixative solution. Cells were then incubated with Cell Event Senescence Green probe (Thermofisher Scientific, Waltham MA, USA) at 1:500 dilution for 2 hours at 37°C with no CO2. Cells were then washed with PBS, 2% FBS, resuspended in FACS staining buffer and data acquired and analyzed by MACSQuant analyzer machine and MACSQuantify software (Miltenyi Biotech, Auburn, CA, USA).