Phase contrast microscopy

When cells were confluent, they were detached by trypsin and re-plated in multiwell 24-well plates. After 24 h the NPs (PNP, loaded-PNP, grafted-PNP) were added in OEC cultures at different concentrations (0.1, 0.5, 1.0 e 5.0 mg/mL) and incubated at 37 °C in DMEM/FBS medium 24 h, for cell viability test. Some OECs were grown without the presence of PNPs, and they were considered as controls. The OEC morphology in all conditions was monitored and the images were captured by phase-contrast microscopy (Zeiss, Oberkochen, Germany) using a 20× lens.
After 24 h that the NPs placed inside OEC cultures, cellular viability was evaluated by the 3-[4,5-dimethylthiazol-2-yl)-2,5-diphenyl] tetrazolium bromide (MTT, Sigma, Milan, Italy) reduction assay, a quantitative colorimetric method was utilized to evaluate cellular cytotoxicity: MTT was added to each multiwell and placed for 2 h in a CO₂ incubator. We removed DMEM/FBS and added MTT solvent (acid-isopropanol/SDS), the cells were shaked for 15 min. A multisKan reader at 570 nm was used to measure the absorbance [23 (link)]. The collected data were expressed as the percentage MTT reduction in comparison with control cells. Differences between OECs grown in presence or not of NPs were assessed using one-way analysis of variance (one-way ANOVA) followed by post hoc Holm–Sidak test.

For the neutralization antibody assay, 96-well plates (TPP) were prepared to obtain monolayers of Vero cells using the procedure described in the cytotoxicity assay section. Antibodies used in these assays were obtained from serum samples collected from CDI-recovered patients included in the SERODIFF study. In order to confirm that the recombinant toxins were also susceptible to the neutralizing activity of these antibodies, 75 µL of a 4 µg.mL⁻¹ solution of toxins was mixed with 75 µL of 1/10 to 1/160 dilutions of serum from CDI-recovered patients and incubated at 37 °C for 60 min [27 (link),28 (link)]. The mixtures were then added to monolayers of Vero cells, and the plates were incubated in 5% CO₂ at 37 °C for 18 h. A negative control (cells in DMEM and 0.1% of BSA only) and positive controls (4 µg.mL⁻¹ and 0.8 µg.mL⁻¹ of rTcdA or rTcdB alone) were also added. Cell morphological alterations were observed under phase contrast microscopy (Zeiss, Oberkochen, Germany).

A. amylolyticus (strain MR3C^T =ATCC BAA-872^T) was grown in Y_N DSM medium containing the following components: yeast extract 0.6% w/v, NaCl 0.6% w/v, and Agar 1.8% w/v (for plate preparation) at 60 °C. The pH of the medium was adjusted at 5.6 by using 0.2 M HCl [1 (link)]. C4-HSL standard was added to the medium at a concentration of 10 µM, and CIN was added to the medium to give final concentrations of 1 and 3 mg mL⁻¹ (7.5 and 22 mM, respectively). Growths were carried out for 24 h and monitored both by evaluating their optical density (O.D., A_540nm) and by observation at phase-contrast microscopy (Zeiss). Moreover, cell viability was checked by means of a serial dilution plating method.

Briefly, 1 × 10³ cells/cm² from dissociated gliospheres at G13 were plated in ultralow attachment six-well plates (Corning, USA) containing CM. The experiment was conducted for 7 days, and cells growing in normal GSM were kept as a control. CM was added every 48 h, and changes in the sphere-forming ability of GSCs were captured by phase-contrast microscopy (Zeiss, Germany).

After mixing equal numbers of plus and minus gametes (2×10⁷ cells/ml in N-free medium) for 10 min, cells were fixed by adding an equal volume of 5% glutaraldehyde. Ciliary adhesions were disrupted by gently pipetting cells 10 times with a 1 ml pipette tip. The numbers of cells in pairs per 200 cells for each sample were determined using phase-contrast microscopy (Zeiss).