Chapter 2 │ Page 41 used for transduction, with multiplicity of infection of 3 (MOI=3), and >1x106 transfection units (TU)/ml, and puromycin was added to select for successfully transduced cells. In order to maintaining selection of fluorescently-transduced cells, the antibiotic, puromycin, was added every 2 weeks to the cell culture. Puromycin quickly kills eukaryotic cells that do not contain this puromycin selection marker (i.e., parental cells or unsuccessfully transduced cells). The addition of puromycin was stopped at least 7 days before the start of any experiment, and fresh medium was added, to circumvent any confounding e ects of the puromycin on our experimental results. This method is common practice and the recommended method for antibiotic selection [24, 25]. Following the transduction and selection process, stable red fluorescent cells (with the mKate2 fluorophore) were obtained (Figure 1a). The lentivirally-incorporated mKate2 fluorophore is the next generation of far-red fluorescent proteins, with excitation/emission maxima at 589 and 633 nm [26]. Freshly thawed cells from liquid nitrogen (PL) and cells with a high passage (PH), minimum of 10 extra culture cycles, from both parental (SC263) and transduced cell lines (SC263T) were seeded into 96-well plates for experiments and incubated for 24 hours before NTP treatment. Strict culturing methods and protocols were implemented to maintain an identical age between the young and older counterparts of SC263/T, respectively. This includes having exactly 2 culture passages (Px+2) over the three required replicates of each experiment. For young cell conditions, this implies experimental initiation at P2, with all experiments performed at P2+2. Once the cells exceeded 5 culture cycles, they were no longer used for the low passage number group. For the aged cellular counterparts, a minimal of 10 additional cell passages was incorporated, meaning P15 at the start of the experiment, and all experiments were completed within two additional passages (P15+2) (Figure 2).
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