Immunocytometry system

For cell cycle analysis, 200,000 cells/well were plated in six well plates, starved for 24 h in medium with 0.4% BSA, transfected with a specific antagomiR and cultured for additional 24 h in medium containing 1% FBS. Then, cells were collected, centrifuged, washed in PBS, stained with a propidium iodide solution and analyzed by flow cytometry using the FACSCalibur Becton Dickinson Immunocytometry System [1] (link). For cell proliferation analysis, 5000 cells/well were plated in 96 well plates, starved for 24 h in DMEM/F12 0.4% BSA and transfected with PL501 or with an irrelevant plasmid as described above. Cells were cultured for further 24, 48 and 72 h in DMEM/F12 1% FBS in presence or absence of rapamycin (500 nM), and the proliferation was calculated by direct cell counting after trypan blue staining, using a Burker chamber [3] (link). Cell survival was measured by the CellTiter cell proliferation assay (Promega, Italy), a method based on the quantitation of a colored compound released by cells in culture medium. Color intensity, directly proportional to the living cells, was detected by a plate reader recording the absorbance at 490 nm [8] (link).

The cell cycle arrest was investigated by the modified protocol of Thangam et al., [17 (link)]. The cells (1 × 10⁶) were cultured in a tissue culture dish and keep them to mature all-night. The cells were treated for 72 h and cells without AgNPs kept as control. After attained at 75% of confluence, the cells were trypsinized and collected in appropriate centrifuge tubes. Then centrifuged at 2500 rpm for 5 min at RT (room temperature) and cells in the pellet were re-suspended in 300 μL of PBS–EDTA to which 700 μL of chilled 70% ethanol was added drop-wise with slow mixing. The solution was added to assure absolute combination of ethanol, and the samples were stored at 0 °C overnight. Subsequently, 1:100 volumes of 20 mg/mL RNase were added to remove RNA contamination, and the mixture was incubated at 37 °C for 1 h. Propidium iodide was added to a final concentration of 50 μg/mL and incubated for 10–20 min at room temperature in dark. The stained cells were analyzed for DNA histograms and for cell cycle phase distribution using flow cytometry (Becton Dickinson Immuno cytometry System, USA)