Fb 1001 500

Human male neonatal foreskin primary fibroblasts (HF043, Dundee CELL products) were seeded at 8 × 10³ cells/cm² in sterile filter-cap flasks (Greiner CELLSTAR) or ~ 1000 cells per well in 384 well plates and cultured in DMEM without phenol red (Gibco 31053-028/31053-044) supplemented with 10% FBS (Biosera, FB-1001/500) and 4 mM l-Glutamine (Sigma Aldrich). All cell incubation (including SASP and viability assays, below) was conducted at 37°C in a humidified incubator at 5% CO₂. No antibiotics were used; mycoplasma negative status was confirmed by regular testing by PCR (Biological Industries EZ PCR Mycoplasma test kit, Geneflow K1-0210). Cells were monitored using an EVOS digital microscope (Life Technologies) and harvested at ∼ 80% confluency using TrypLE Express (Invitrogen, 12604021). After harvesting, cells were resuspended in DMEM with FBS and 20 µL of a homogenous suspension was counted using a Cellometer T4 (Nexelcom); both cell number and cell diameter in suspension were recorded. Population doublings (PD) were calculated using the formula:

MRC5 and HEK293 cells were grown in Dulbecco’s Minimum Essential Media (DMEM, Sigma-Aldrich) supplemented with 10% fetal bovine serum (FBS) 1% penicillin/streptomycin (Sigma-Aldrich) and 1% l-Glutamine (Sigma-Aldrich). Human primary fibroblasts were grown in DMEM (Sigma-Aldrich) supplemented with 10% fetal bovine serum (Biosera, FB-1001/500), 1% penicillin/streptomycin (Sigma- Aldrich), 1% l-Glutamine (Sigma-Aldrich) and 1% non-essential amino acids (Sigma-Aldrich).