7 aad staining solution

Cell clones were plated in 6-well plates at an initial density of 2 × 10⁴/cm² in two triplicate sets (for −/+tetracycline) and left to adhere overnight. Growth medium was replaced the following day, and induction was performed with 1 μg/ml tetracycline (Sigma Aldrich, Dorset, UK). After 48 hrs cells were harvested with 0.05% Trypsin (Gibco, MA, USA) and pelleted at 200 × g for 5 mins at room temperature. 5 × 10⁴ cells were washed in PBS, fixed and permeabilized in 2 ml of ice-cold 70% ethanol followed by incubation at −20 °C for at least one hour. Cells were pelleted as before, washed twice in PBS and resuspended in 50 μl of 50 μg/ml 7-AAD staining solution (BD, New Jersey, USA), supplemented with 0.1% Triton X-100 (Sigma Aldrich, Dorset, UK), 2.5 units of RNase A (Thermo Scientific, MA, USA). Cellular DNA was stained for 30 mins at room temperature while protected from light. Samples were diluted with PBS immediately before collecting area fluorescence measurements on a linear scale with an Attune flow cytometer (Invitrogen, Paisley, UK). Data were analyzed using FlowJo software (TreeStar, OR, USA) where cell cycle gates (Sub G1, G1, S and G2/M phases) were set on the uninduced (no tetracycline) sample of each clone.

Cell proliferation assays were performed by directly measuring DNA synthesis using an Alexa Fluor 647 click-iT plus EdU flow cytometry kit (Thermo Fisher) according to the manufacturer’s instructions. 5-Ethynyl-2′-deoxyuridine (EdU, 10 μM) was added to the culture medium for a 12-h incubation in a humidified atmosphere at 37 °C with 5% CO₂ at the end of the 12-h coincubation (0, 10, 20, 40, 80 µg/mL HcESPs with T cells) period. Subsequently, T cells were fixed with 4% paraformaldehyde in PBS and permeabilized with Click-iT saponin-based permeabilization and wash reagent, followed by Click-iT reaction to couple EdU with the Alexa Fluor 647 dye. After two washes with 3 mL of 1% BSA in PBS, T cells were treated with 7-AAD Staining Solution (BD Biosciences), and standard flow cytometry methods were used to determine the percentage of S-phase cells in the population. Each experiment consisting of three replicates was performed in triplicate.