Quantifying p16 and p53 Expression in PBMCs

p16 and p53 expressions were determined by flow cytometry as described in [27 (link)]. Briefly, cryopreserved PBMCs were resuspended in a PBS solution supplemented with 10% fetal bovine serum, washed with PBS 3% fetal bovine serum (FBS) and fixed using the Cytofix-CytopermTM kit (BD Biosciences, Franklin Lakes, NJ, USA). Fixed cells were incubated with anti-human p16 (BD Pharmingen, Franklin Lakes, NJ, USA) and anti-human p53 (BD Pharmingen, Franklin Lakes, NJ, USA) antibodies for 16 h at 4 °C. The secondary antibody was Alexa 488 anti-rabbit (Thermo Fisher Scientific, Waltham, MA, USA) 1:1000 in PBS 3% FBS for 1 h at room temperature. Quantification was performed using a Cytoflex (BD Biosciences, Franklin Lakes, NJ, USA) flow cytometer and FlowJo software v7.6.1, considering a cell population of 100,000 cells for each analysis.