Macsquant analyzer machine

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).

FACS Staining Buffer (Thermofisher Scientific, Waltham MA, USA) was used to stain cells on ice. FC receptors (FcR) were blocked using mouse FcR blocking reagent (Miltenyi Biotec, Auburn, CA, USA) for 15 minutes protected from light followed by incubation with conjugated antibodies on ice for 30 minutes. Cells were then washed and re-suspended in FACS buffer and data was acquired using MACSQuant analyzer machine and MACSQuantify software (Miltenyi Biotech Auburn, CA, USA). Antibodies used: CD11c APC; clone N418 (Affymetrix, eBioscience, Thermofisher Scientific, Waltham MA, USA)., CD86 (B7-2) PE; clone GL1(Affymetrix, eBioscience, Thermofisher Scientific, Waltham MA, USA)., CD80 FITC; clone 16-10A1(Invitrogen, Thermofisher Scientific, Waltham MA, USA), CD4 Vioblue; clone GK1.5 (Invitrogen, Thermofisher Scientific, Waltham MA, USA), MHCII Viogreen; clone M5/114.15.2, (Miltenyi Biotech Auburn, CA, USA).

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).