Pi propidium iodide

P3 BMSCs were seeded at a cell density of 10⁵ cells/ml in a 6-well plate coated with poly-L-lysine. After cells adhered to the surface, the cells were processed via serum starvation as follows. The culture medium was changed to DMEM containing 10, 5, 1, 0.5, 0.1, and 0% FBS and cultured in an incubator for 12, 24, 36, 48, and 72 hours, respectively. Next, the cells were rinsed with 0.1 M PBS three times. The adherent cells were digested with 0.25% trypsin into a cell suspension and collected into an EP tube, where the cells were fixed with 70% ethanol for 2 hours. After PI (propidium iodide) (Sigma-Aldrich, USA) staining for 10 s, a flow cytometer (Beckman, EPICS XL coulter) was used to detect the percentage of G0/G1, G2/M, and S cells for every 10⁴ cells. The experiment was repeated five times for each group.

S-phase cells were detected using an FITC/BrdU (bromodeoxyuridine) staining kit (BD Biosciences) according to the manufacturer's instructions. For all cell lines used, BrdU was incubated for 30 min at 37°C. Alternatively, DNA content was measured using PI (propidium iodide; Sigma–Aldrich) staining as described previously [3 (link)]. Cell viability was measured by the ability of live cells to exclude PI as described in [3 (link)]. For all samples, 10000 cells were counted using a FACSCalibur instrument (BD Biosciences) and analysed using the provided software. For both BrdU-stained and PI-stained DNA content measurements, multi-cell-aggregates as well as sub-G₁ cells were discarded for the analysis and G₀/G₁, S (when applicable) and G₂/M populations were expressed as the percentage of total live cells (without sub-G₁). FACS data are presented as means±S.D. for three biological experiments.

Anticancer agents interfere with various cell cycle phases. The cancer cell cycle may help elucidate the DNA-binding copper(II) complex mechanisms. HepG2 cells were inoculated onto six-well plates at 1 × 10⁵/well at 37 °C overnight under a 5% CO₂/95% O₂ conditions. The cells were left untreated or subjected to complex 1 or complex 2 for 24 h, trypsinized, rinsed with cold PBS (phosphate-buffered saline, Hyclone Laboratories Inc., Logan, UT, USA), fixed using 70% (v/v) ethanol, and kept at 4 °C. Afterwards, the pellets were washed twice with 1.0 mL PBS. Then, 10.0 mL of 20 μg/mL RNase (Sigma-Aldrich Corp., Shanghai, China) and 10 μL of 50 μg/mL PI (propidium iodide, Sigma-Aldrich Corp., Shanghai, China) were introduced, and the cell suspensions allowed to grow for 0.5 h at 37 °C. The samples were then analyzed with a Beckman CyAn-ADP flow cytometry platform (Beckman Coulter, Brea, CA, USA). Ten thousand cells were analyzed per sample. A similar procedure was used to assay the HepG2 cell cycles after 48 h treatment with complex 1 or complex 2.

HUVECs were cultured in a hypoxia incubator and synchronized in the G0/G1 phase of the cell cycle, which was treated for 24 hours with gradient concentrations of FA (0.1 μg/ml, 1 μg/ml, and 5 μg/ml). At end of the period, cells were digested and washed three times with cold PBS (Beyotime technology, Shanghai, China), and then, the cell concentration was adjusted (1 × 10⁵/ml). Then, 70% ethanol was applied to fix cells overnight. In the end, the cells were stained with PI (propidium iodide) (Sigma-Aldrich, St. Louis, MO, USA) for 10 min at 4°C (notice to avoid light). Fluorescence intensity was detected using a flow cytometry analyzer (BD Biosciences, San Jose, USA). The analysis of percentages of every phase (G1, S, and G2 phase) was accomplished with the GraphPad software.

For dual staining, 100 µL of the bacterial culture of E. coli DH5α and S. aureus ATCC6538P (bacteria were grown to mid-logarithmic phase) was incubated in the dark for 2 h at 37 °C in agitation in the presence or absence of F. vulgare subsp. vulgare var. vulgare EO, a concentration of 200 µg/mL. After the incubation, 10 µL of bacterial culture was mixed with DAPI solution (40, 6-diamidino-2-phenylindole dihydrochloride; Sigma Aldrich, Milan, Italy) (1 µg/mL DAPI final concentration) and PI (propidium iodide; Sigma Aldrich, Milan, Italy) 20 µg/mL. Samples were observed using an Olympus BX51 fluorescence (Olympus, Tokyo, Japan) using a DAPI filter (excitation/emission: 358/461 nm) [46 (link)].

HCT 116^p53−/− cells, uL3ΔHCT 116^p53−/− and eL8∆HCT 116^p53−/− cells were seeded into 60 mm tissue culture plates at confluency of about 50%–60%. Then, cells were treated with LQ1 at 10 μM. After 48 h, the cells (2 × 10⁶) were harvested and centrifuged at 400× g for 5 min, washed once with cold PBS (Dulbecco’s phosphate-buffered saline, Sigma-Aldrich, St. Louis, MO, USA) and resuspended and fixed by adding 0.5 ml ice-cold 70% ethanol dropwise. Then, the cells were incubated on ice overnight. The cells were spun down and washed twice with PBS. They were resuspended in 500 μl of PBS and incubated with 200 μg/mL RNAse (Sigma-Aldrich, St. Louis, MO, USA) for 30 min at 37 °C; then, 50 μg/mL PI (Propidium Iodide, Sigma-Aldrich, St. Louis, MO, USA) was added and cell lysate was incubated for 30 min protected from light. Cell cycle distribution was analyzed using BD FACS CantoII Cytometer (BD Biosciences, San Jose, CA, USA) and Diva software (v6.x).