Cytomics fc 500 mpl system

Apoptosis was evaluated using the Dead-End^TM Fluorometric TUNEL System (Promega, Madison, WI, USA). Cells were fixed with 2% paraformaldehyde and permeabilized with 66% ice-cold ethanol for 2 h at 4 °C. Subsequently, the cells were rinsed with PBS and incubated in equilibration buffer for 5 min at RT. Fixed cells were incubated with fluorescein-12-dUTP in a reaction catalyzed by recombinant terminal deoxynucleotidyl transferase (TdT) for 1 h at 37 °C. After washing, cells were incubated with propidium iodide (PI)/RNase Staining Solution (Cell Signaling Technology, Danvers, MA, USA) for 30 min at RT in darkness. Stained cells were analyzed by flow cytometry using a Cytomics FC 500 MPL system and quantified with MXP software (both from Beckman Coulter Brea, CA, USA).

After 3-day in culture, E3330 treated and untreated DPCs (1 × 10⁶) were respectively collected by exposure to trypsin/EDTA for 1 min and centrifuged at 1000 rpm for 5 min. Cell precipitates were washed twice with 0.01 mol/L PBS containing 2% FBS and resuspended in 1 mL physiologic saline, fixed in 500 μL cold 75% alcohol, and stored at 4 °C overnight. Then, each sample was washed again with PBS, and incubated with RNase at 37 °C for 30 min and then added with propidium iodide (100 mg/mL; Keygentec, Nanjing, China) at 4 °C for at least 30 min. Cell cycle fractions (G0G1-, S-, G2M-phases) were then determined by Beckman Coulter Cytomics FC 500 MPL system (Beckman Coulter, USA). Three independent experiments from different donors were performed for each group.