Evos digital microscope

HF043 diploid neonatal primary human fibroblasts were obtained from Dundee CELL products, while HeLa cells were a gift from Prof. F. Barr (University of Oxford). Cells were routinely subcultured in DMEM (Sigma) with 10% FCS (Gibco) and incubated in a humidified incubator at 37 °C with 5% CO₂. Cells were imaged using an EVOS digital microscope (Life Technologies) and routinely sub-cultured according to standard protocols (Walters et al. 2016 (link)). For diameter analysis and calculation of proliferation rates by cell counting, 20 µl of cell suspension was taken during cell harvesting for analysis using a Cellometer T4 (Nexelcom). The primary cells used in all experiments were at low CPD (cumulative population doubling, < 50 CPD c.f. HF043 fibroblasts reach replicative senescence around CPD 90 (Walters et al. 2016 (link))).

Human neonatal foreskin fibroblast line HF043 (Dundee CELL products) was cultured in DMEM (Sigma) supplemented with 10% fetal calf serum (Gibco) in the absence of any added antibiotics, at 37°C in a humidified incubator with 5% CO₂. Cells were monitored microscopically using an EVOS digital microscope (Life Technologies) and harvested when ∼80% confluent using TrypleExpress (Invitrogen). Following resuspension in DMEM with FCS, 20 μl of the cell suspension was counted and cell diameters measured using a Cellometer T4 (Nexelcom). Cells were seeded at 2×10⁵ per T25 flask (Greiner), or at 1×10⁴ in 24 well plates (Greiner). Population doublings (PD) were calculated as:
$\mathit{\text{PD}}=\frac{{\log }_{10}(\mathit{\text{total}} \mathit{\text{cells}} \mathit{\text{harvested}} / \mathit{\text{total}} \mathit{\text{cells}} \mathit{\text{seeded}})}{{\log }_{10}2}$
Cumulative population doublings (CPD) were calculated as the sum of PD values.
AZD8055 (Selleckchem) was reconstituted to 1mM in DMSO and stored in aliquots at −20°C protected from light. Prior to drug treatment, medium was removed, cells washed with PBS, then fresh medium supplemented with drug was added. Total volume of drug or DMSO added to the culture medium never exceeded 1:10,000 v/v. Doses of 35 nM and 70 nM were chosen as effective but non-toxic based on our preliminary studies (not shown).