Culturing of Peripheral Blood Mononuclear (PBMCs) and HEK Cells

PBMCs were isolated using the Ficoll Hypaque (GE Healthcare Life Sciences, Boston, United Kingdom) density gradient and stored in liquid nitrogen. PBMCs were thawed and maintained in culture flasks containing RPMI (GIBCO, Massachusetts, United States) and 10% AB* serum (Sigma-Aldrich, Steinhein, Germany) for 24h. Subsequently, PBMCs were cultured in a 24-well plate under unstimulated conditions (medium only), and PHA-stimulated (25 μg/mL, Sigma-Aldrich, Steinhein, Germany) conditions for three days. Afterwards, 2,000,000 cells were collected in nuclease-free 1.5 mL-microfuge tubes. Cells were washed with PBS without calcium or magnesium and pelleted by centrifugation at 8000 x g for 5 min at room temperature (37°C). The supernatant was carefully discarded to ensure that all residual liquid was removed, and the pellet was used for cell lysis.

HEK 293 cells were used as positive controls as this cell line overexpresses telomerase (36). HEK cells were cultured in culture flasks (25 and 75 cm³) using DMEM (GIBCO, Massachusetts, United States) supplemented with 10% fetal bovine serum (FBS) (GIBCO, Massachusetts, United States) until they became 85% confluent (Figure 1). Subsequently, the cells were split into two culture flasks for further growth until a count of 1x10⁸ cells was reached. HEK cells were then pelleted following the same procedure described for the PBMCs.