Propidium iodide rnase solution

Cells were cultured with vehicle or 15 μmol/L pentamidine in 6-well plates for 24 h. After culture, cells were harvested, washed with phosphate buffer saline (PBS), and then suspended in propidium iodide/ RNase solution (BD Biosciences, San Jose, CA, USA). Finally, the cells were analyzed using a Becton–Dickinson FACS Vantage flow cytometer (Becton Dickinson, San Jose, CA, USA).

Flow cytometry analysis was performed using the BD Cytofix/Cytoperm Fixation/Permeabilization Kit (554714, BD Biosciences). For quantification of OCT4 transcription factor (also known as POU5F1), NBFR-FF-bESCs were permeabilized and blocked in Perm/Wash Buffer supplemented with 2% v/v of NDS for 15 min at room temperature. Cells were then incubated with anti-POU5F1 (1:500; sc-8628, Santa Cruz Biotechnology) primary antibody for 1 h at room temperature and washed with Perm/Wash Buffer. Secondary antibody incubation was performed for 30 min at room temperature with donkey anti-goat IgG Alexa Fluor 488 antibody (1:500; A-11055, Invitrogen) protected from light. Cells were kept at 4 °C until flow cytometry analysis was performed.
Cell-cycle analysis was performed fixing NBFR-FF-bESCs with ice-cold 70% ethanol and stained using Propidium Iodide/RNase Solution (550825, BD Pharmingen). All data were acquired from two independent NBFR-FF-bESCs lines and analyzed in a FACScan flow cytometer (Becton Dickinson) equipped with a 488 nm excitation laser using the CellQuest Pro Software (Becton Dickinson).