Propidium iodide flow cytometry kit

Cell cycle analysis and apoptosis rates following VIIb treatment were investigated using a Propidium Iodide Flow Cytometry Kit (ab139418; Abcam, Cambridge, UK) [60 (link)] and Annexin V-FITC apoptosis kit (Catalog: K101-25; BioVision Research Products, San Francisco, CA, USA) [61 (link)], respectively, following the manufacturer’s instructions as stated by a previous study [17 (link)].

Cell cycle assay was studied to detect the divisional phases of the cells, and to do this DNA content is utilized. The percentages of untreated SKOV3 cells and treated SKOV3 cells (treated for 24 h with an IC₅₀ value of SnO₂ NPs and FA-SnO₂ NPs) through the different phases of the cell cycle were evaluated using a flow cytometry kit (Abcam, Propidium Iodide Flow Cytometry Kit, catalog ab139418) as previously mentioned [45 (link),46 (link)]. Briefly, SKOV3 cells were seeded and incubated overnight then a cell density of about 1 x10⁶ cells were placed in a 6-well culture plate for 24 h. After that, these cells were treated with SnO₂ NPs and FA-SnO₂ NPs for 24 h. Subsequently, the cells were trypsinized, harvested, and overnight fixed in ice-cold ethanol (70%). Furthermore, the cells were centrifuged, washed with PBS twice, treated with RNase and then added to PI solution (40 μM ml-1 in PBS) for 30 min in the dark at 37°C for staining the control and treated cells. Finally, the percentages of cells existing in each cell cycle phase (sub-G1, G0/G1, S, and G2/M phases) were measured using DNA quantification using flow cytometry and the obtained results were displayed in the form of a histogram.

The effect of F2 fraction on cell cycle distribution was assessed by flow cytometry after staining of HaCaT-ras A5, and HaCaT-ras II4 cells with propidium iodide (PI). Briefly, both cells lines (1x10⁵cells/mL) were treated with different concentrations (25 and 50 μg/mL) of F2 fraction and cultured in 6-well plates for 48 h. The treated cells were harvested, washed with PBS and fixed with 70% ethanol on ice. The cells were then washed with cold PBS, suspended in 200 μL 1× Propidium Iodide + RNase staining solution and incubate at 37 °C in the dark for 30 min. Propidium Iodide Flow Cytometry Kit (Abcam, Cambridge, UK) was used for cell cycle analysis. The DNA content of the cells was measured by C6 flow cytometer (BD Accuri Cytometers, Ann Arbor, MI USA) and the population of each phase was determined using CFlow Plus analysis software (BD Accuri Cytometers, Ann Arbor, USA).

C4-2B cells were co-transfected with miR-221 and miR-222 precursors or inhibitors (Ambion) at a final concentration of 20 nM each using Lipofectamine RNAiMAX Transfection Reagent (Life Technologies) according to the manufacturer’s instruction. Cell number was counted 3 days after transfection. Cell cycle analysis was performed in parallel using Propidium Iodide Flow Cytometry Kit (Abcam). Cell cycle distribution of 10,000 gated cells is presented.