Fitc conjugated annexin 5 and 7 aad

Single cell suspension was prepared using trypsin-EDTA (0.25%). For apoptosis assay, cells were suspended in 1× binding buffer (BD Biosciences, USA) and incubated with FITC-conjugated annexin V and 7-AAD (BD Biosciences) for 25 min at RT. Cells were analyzed using BD FACSVerse (BD Biosciences). For the detection of superoxide and reactive oxygen species (ROS), cells were incubated with MitoSOX Red mitochondrial superoxide indicator (Thermo Fisher Scientific) for 10 min at 37°C and analyzed using BD FACSVerse (BD Biosciences). Data were analyzed using the FlowJo software (ver. 10).

Cells were washed and seeded at a density of 150,000 cells/ml in the presence or absence of 40 µM of 3-deaza-uridine or 200 µM of cytidine in 24 well plates (one well per time point). At each time point, the cells were stained with FITC-conjugated annexin V and 7-AAD according to the manufacturer’s instructions (BD Biosciences) and analysed using a LSRFortessa X-20 cytometer and FlowJo software (RRID:SCR_008520; BD Biosciences). HEK cells, maintained with 200 μM of cytidine, were washed, detached using trypsin–EDTA (Life Technologies), and seeded in six-well plates at a density of 26,000 cells/cm² in the presence or absence of 200 μM of cytidine in DMEM, 10% FBS, 1% penicillin–streptomycin. At each time point, the cells were imaged using an Axio Vert A1 microscope (Zeiss) at the indicated magnification. The imaged cells were then detached using trypsin, mixed with their supernatant, and stained as described for the Jurkat cells. With the exception of the CTPS1+2-KO cells that never reached confluency, all the other cell populations were passaged (seeding of 26,000 cells/cm²) in parallel when they approached or reached confluency and maintained under the same culture conditions.