Rnase solution

The cell cycle was evaluated by cytometry using propidium iodide (PI) solution (Sigma-Aldrich). After fixation in 70% ethanol solution, cells were washed twice in PBS and stained using 250 μl of RNase solution (2 mg/ml, Sigma-Aldrich) added to 250 μl of PI solution (0.1 mg/ml in 0.6% Triton-X in PBS) for 45 minutes in the dark at room temperature. Cells were then transferred through capped tubes to avoid clumps during fluorescence detection. Samples were kept on ice and protected from light until cytometry analysis (Accuri C6, BD Biosciences).

For cell cycle analysis 500,000 cells were treated for the indicated time with doxycycline 2μg/ml, collected and fixed with ice cold 70% ethanol. After O/N fixing at −20°C, cells were washed in PBS, resuspended in 50μl of 10μg/ml RNase solution (SIGMA) and incubated for 10 minutes at 37°C. 500μl of staining solution (50μg/ml propidium iodide in PBS) was added to the cells, followed by additional incubation 30 minutes at 37°C. Stained cells were analyzed by flow cytometry and at least 10,000 cells per sample were collected. Data were analyzed using CELLQuest acquisition/analysis software.

Tni-FNL and Sf9 cells in exponential growth phase were harvested at a cell concentration of approximately 1 × 10⁶ cells/mL and centrifuged at 1050 rpm for 5 min after which the supernatant was discarded. The cell pellet was suspended in 20 mL of cold (−20 °C) 70% ethanol for fixation and the samples were stored at −20 °C. On the day of flow cytometry analysis, cell samples were centrifuged at 1050 rpm for 5 min to remove the ethanol fixative. The supernatant was discarded and the cells were washed two times with 10 mL phosphate buffered saline (PBS). The sample was centrifuged again at 1050 rpm for 5 min, the supernatant was discarded, and the pellet was suspended in 1 mL of RNase solution (250 μg/mL; Sigma, St. Louis, MO, USA) for 20 min at 37 °C. A 50 μL aliquot of propidium iodide (PI; 50 μg/mL) was added to each sample, mixed, and incubated at room temperature for 5 min, before analysis by flow cytometer.

The effect of DEX on the expression and nuclear localization of CAR were evaluated in vitro in a human cell line of hepatoblastoma cells (HepG2), according to the protocol described in detail in Castellani et al. [29 (link)]. Briefly, HepG2 cells were seeded in a 24-well plate with glass coverslips. After 24 hours, cells were pre-treated either with 0.2 or 2 μM DEX for 4 hours and then with 1 ng/mL LPS for 24 hours. The effect of DEX was evaluated using untreated and LPS-treated cells as negative and positive controls, respectively. At the end of the treatment, cells were fixed in 4% paraformaldehyde and incubated with a rabbit polyclonal primary antibody antiCAR (1:100 dilution, Abcam, Cambridge, UK) for 1 h, using an Alexa Fluor 488 anti-rabbit as secondary antibody (1:500 dilution, Jackson ImmunoResearch Europe Ltd., Ely, UK). For nuclear staining, cells were incubated with DAPI (100 pg/mL, Life Technologies, Monza, Italy), after treatment with a 2 mg/mL RNAse solution (Sigma-Aldrich, Milan, Italy). The images of the immunostained cells were acquired by means of a confocal microscope Zeiss LSM 800, using a 63X magnification. ImageJ software was used to quantify the intensity of the fluorescent signal and the co-localization between the nuclear marker DAPI and CAR signal.

For cell cycle analysis 500,000 cells were treated for the indicated time with doxycycline 2μg/ml, collected and fixed with ice cold 70% ethanol. After O/N fixing at −20°C, cells were washed in PBS, resuspended in 50μl of 10μg/ml RNase solution (SIGMA) and incubated for 10 minutes at 37°C. 500μl of staining solution (50μg/ml propidium iodide in PBS) was added to the cells, followed by additional incubation 30 minutes at 37°C. Stained cells were analyzed by flow cytometry and at least 10,000 cells per sample were collected. Data were analyzed using CELLQuest acquisition/analysis software.

AGS cells (10⁶ cells/mL) were incubated in fetal calf serum (FCS)-free F-12K medium at 37 °C for 24 h and then synchronized. The medium was replaced with F-12K medium containing FCS, and then the AGS cells were incubated with the culture supernatant of each of the four types of nitrate-reducing bacteria and H. pylori at 37 °C for 24 h. Trypsin was added to the culture plate, from which the F-12K medium was removed, and then the plate was incubated at 37 °C for 10 min. AGS cells were collected in 15 mL tubes, fixed with cold 100% methanol at 4 °C for 2 h, centrifuged at 1000 rpm for 10 min, and then decanted. After washing with PBS, the cells were centrifuged again at 1000 rpm for 10 min and then decanted. This process was repeated twice. The cells were incubated with 0.25 mg/mL RNase solution (Sigma-Aldrich, St. Louis, MO, USA) at 37 °C for 30 min. The cells were incubated overnight with propidium iodide solution (Sigma-Aldrich) at 4 °C.

Around one cm² of young and well-developed leaf from each population together with a half area of leaf material from Glycine max (L.) Merr. cv. Polanka (2C DNA = 2.50 pg) as internal standard were chopped (1 min) into small pieces in 1 mL of woody plant buffer, supplemented with 50 µL of Propidium Iodide (PI, Fluka) as DNA staining agent and 50 µL of RNase solution (Sigma-Aldrich Corporation, MO, USA). Then, the resultant nuclear suspension was passed through a 30 µm Partec green nylon mesh and analyzed using a BD FACSCanto II flow cytometer (BD Biosciences, Bedford, MA, USA), equipped with a 532 nm green high-grade solid-state laser. Analyses were carried out on five (n = 5) individuals per population using a BD FACSDiva^TM Software. Histograms with a coefficient of variation (CV) in the range between 3.20 and 4.50% for G1 peaks were evaluated. The 2C DNA (pg) content was estimated using the linear relationship between the ratio of the G1 peak means of the target population/G₁ peak mean of the internal standard × Standard 2C DNA (pg).