Cell quest acquisition software

Both nontransfected and transfected U87 and U251 cells were harvested by trypsinization, washed three times in cold PBS and fixed in 70% ethanol at 4 °C overnight. After fixation, the cells were washed and resuspended in cold PBS and incubated in a solution of 10 mg/ml RNase and 1 mg/ml propidium iodide (Sigma-Aldrich, St. Louis, MO, USA) at 37 °C for 30 min in the dark. Finally, the DNA content was determined using flow cytometry (BD Biosciences, Franklin Lake, NJ, USA). The percentage of cells in the G0/G1, S and G2/M phases was determined using Cell Quest acquisition software (BD Biosciences).

The cells were plated into 6-well plates and incubated for 1 and 3 days with nutrient solution containing various concentrations of PTX (0, 0.1, and 0.3 nM). Briefly, the cells were collected and fixed in 0°C 70% ethanol and stored at -20°C. The cells were then washed and resuspended in cold PBS and incubated at 37°C for 30 min with 10 mg/mL RNase and 1 mg/mL propidium iodide (Sigma–Aldrich, USA). DNA content analysis was performed by flow cytometry (BD, San Diego, CA, USA). The percentage of cells in different cell cycle phases was determined with Cell Quest acquisition software (BD Biosciences Pharmingen, USA).

MDA-MB-231 cells were treated with various concentrations of Pu-erh tea water extracts (0, 100, and 300 μg/mL) for 24 h, and untreated cells served as a control (0 μg/mL). Then, the cells were collected and fixed in cold 70% ethanol and stored at -20°C. The cells were then washed and resuspended in cold PBS and were allowed to incubate at 37°C for 30 min with 10 mg/mL RNase and 1 mg/mL propidium iodide (Sigma–Aldrich, Germany). DNA content analysis was performed by flow cytometry (BD, LSRFortessa^TM, San Diego, CA, USA). The percentage of cells in different cell cycle phases was determined with Cell Quest acquisition software (BD Biosciences, Pharmingen, San Diego, CA, USA).

ASM cells were cultured in the complete medium with 1,25-(OH)2D3 for 2 h before treated or not with 50 ng/ml of VEGF for 48 h. All the cells were collected, and 1 × 10⁶ cells were centrifuged, resuspended in ice-cold 70% ethanol and stored at −20 °C until further analysis. Washed cells were stained by 0.1% Triton X-100 in 0.01 M phosphate-buffered saline (pH 7.2) with 50 μg/ml propidium iodide (Sigma-Aldrich) and 1 mg/ml RNase A (Invitrogen), and incubated at 37 °C for 30 min in the dark. Samples of the cells were then analyzed for their DNA content using FACScan flow cytometry (Beckman, Miami, FL), and cell cycle phase distributions were analyzed by the Cell Quest acquisition software (BD Biosciences, Franklin Lanes, NJ). All experiments were performed in duplicate and repeated twice.