Fxcycle pi rnase kit

For flow cytometry (FACS)-based analysis of the cellular DNA content, non-infected and infected host cell layers (n = 6) were washed with 1X PBS buffer and detached from the plate by trypsin/EDTA (0.25%) treatments at 37°C for 5 min. Then, cells were pelleted at 400 x g for 5 min. The cell pellet was resuspended, washed in 1X PBS buffer, and centrifuged at 400 x g for 5 min. Thereafter, cells were fixed with ice-cold absolute ethanol for 3 min at −20°C and pelleted again (400 x g, 5 min). Samples were stained with the commercial FxCycle PI/RNAse kit following the manufacturer’s instructions (Thermo Fisher, F10797). The samples were analyzed by an Accuri C6 Plus Flow Cytometer analyzer (Becton-Dickinson, Heidelberg, Germany) applying 535/5 nm excitation and emission collected in a 617/20 band-pass. The cells were gated according to their size and granularity. Data analysis was performed via FlowJo LLC software (Ashland, OR).

Both analyses were performed using FACSCalibur flow cytometer (Becton Dickinson, Warsaw, Poland).
Cell cycle analysis was performed using FxCycle PI/RNase kit (Thermo Fisher Scientific, Warsaw, Poland) and the analysis of apoptosis was performed by dual staining with Annexin V-FITC and propidium iodide (PI) (Thermo Fisher Scientific, Warsaw, Poland) following the manufacturer’s protocol. For both assays, 10⁶ cells/mL suspensions were used. Samples were analyzed using the Cell Quest and FCS Express 6 software.