Fxcycle pi rnase a staining solution

For propidium iodide (PI) staining, treated cells (~1 million per condition) were washed with cold PBS, then fixed in 80% ethanol overnight at −20 °C. After fixing, cells were pelleted, washed with PBS, and reconstituted in 500 μL FxCycle PI / RNAse A staining solution (Thermo Fisher). Cells were stored overnight at 4 °C and analyzed using a BD Fortessa flow cytometer.

The effect of radiation exposure on cell cycle progression was examined using propidium iodide (PI) DNA staining. The fluorescence intensity of PI-stained DNA reflects the DNA content of a cell, which determines the proportion of cells in the different cell cycle phases. Forty-eight to seventy-two hours after radiation exposure, 3 x 10⁶ cells were washed with PBS, and fixed in ice cold 70% ethanol for at least 1h at -20°C. They were then allowed to warm at room temperature, washed with PBS, and resuspended in 0.5 mL of FxCycle PI/RNase A staining solution (Thermo Fisher Scientific, Rockford, IL) for 30 min, following which PI-elicited fluorescence was measured using the MACSQuant Analyzer 10 (Miltenyi Biotec Inc). Each sample was collected as 10,000 events, and the corresponding cell cycle distribution was then determined according to the DNA content on FlowJo software.

Cells were fixed with 70% ethyl alcohol (EtOH) and stained with antibodies against ORF57 (sc-135746, SCBT) and anti-mouse IgG (H+L) Alexa Fluor Plus 647 (A-32728, Thermo Scientific), where indicated. DNA was then stained with FxCycle PI/RNaseA staining solution (Thermo Scientific) for 1 h. The cells were analyzed using a BD Accuri C6 flow cytometer and data were processed using the CFlow Sampler software (BD).
For cell proliferation analysis, cells were maintained for 2 h in medium containing 10 μM 5-ethynyl-2’-deoxyuridine (EdU). Subsequently, the cells were fixed in 4% paraformaldehyde in PBS and the EdU incorporated in the cell DNA was coupled to Alexa Fluor 647 according to the manufacturer’s instructions provided in the Click-iT EdU Alexa Fluor 647 imaging kit (Thermo Fisher Scientific). Images were acquired using a CellInsight High Content microscope (Thermo Fisher Scientific) and analyzed using Cell Profiler 3.0 software to quantify EdU-positive cells.