Fc500 mpl system

Cell apoptosis rates was assessed with the Annexin V-FITC and propidium iodide (PI) apoptosis kit (KeyGEN Biotech.CO., LTD, Nanjing, Jiangsu, China) according to the manufacturer's protocol. After dual-staining with Annexin V-FITC and PI, the cells were immediately analyzed with flow cytometry (FC 500 MPL system; Beckman Coulter, Inc., Miami, FL, USA).

A total of 1×10⁶ cells in PBS were stained with FITC-Annexin V and PI (BD Biosciences) in accordance with the manufacturer’s instructions. Then, the apoptotic analysis of the samples was measured using flow cytometry. Data acquisition and analysis were performed on an FC 500 MPL System (Beckman Coulter, Inc., CA, USA).

HeLa cells were regularly cultured in DMEM/F-12 medium supplemented with 10% fetal bovine serum, 100 IU/ml penicillin and 100 μg/ml streptomycin in a humidified incubator supplemented with 5% CO₂ at 37°C. Synchronizing cells to different phases referred to established protocols (37 (link)–39 (link)). Generally, G1-phase enriched cells were obtained by treating cells with 400 μM L-mimosine (Sigma) for 20–24 h. To achieve a maximum S-phase population, cells were blocked with 2 mM thymidine (Sigma) for 20 h and released in fresh medium for 10 h, followed by 20 h of blocking with thymidine and finally culturing in fresh medium for another 4 h. Cultures containing G2-phase arrested cells were obtained by incubating with fresh medium containing 0.1 μg/ml KaryoMAX^® Colcemid (Life Technologies) for 6 h after two rounds of thymidine blocking. Flow cytometry was used to assess cell cycle distribution. Cells were fixed with 70% ethanol and then stained with 20 μg/ml propidium iodide (PI) solution containing 0.1% Triton X-100 and 200 μg/ml RNase A, referring to standard protocol (40 (link)). Cell cycle distribution, through quantifying the PI fluorescence intensity, was determined in a FC500 MPL system (Beckman Coulter). At least 15 000 cells were collected for each condition and the generated cell cycle histograms were analyzed with Watson pragmatic model in Flowjo software package.