Anti human p16

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.

Flow cytometry was performed from cryopreserved PBMCs. Cells were resuspended in a PBS solution supplemented with 10% fetal bovine serum. PBMCs were washed with PBS 3% fetal bovine serum (FBS) and fixed using the Cytofix-CytopermTM kit (BD Biosciences). The antibodies used were anti-human p16 (BD Pharmingen) and anti-human p53 (BD Pharmingen), and the secondary antibody was anti-rabbit Alexa 488 (Thermo Fisher Scientific, 1:1000). Cells were incubated with a specific antibody for 16 h at 4 °C, and then, the samples were washed twice with PBS 3% FBS. The secondary staining was performed in PBS 3% FBS for 1 h at room temperature and washed twice with PBS 3% FBS. Finally, the analysis was performed using a Cytoflex (Beckman Coulter) flow cytometer and FlowJo software v10.0.10. The cell population considered by each sample for the analysis was 100,000 cells.