Platereader af2200

The cytocompatibility of the previously selected fractions of CDs were assessed on human breast cancer cell lines (MDA-MB-231 by Sigma Aldrich, Milan, Italy) by MTS assay (Promega). Cells were seeded on a 96 multiwell plate at a density of 1.5 × 10⁴ cells/well (200 μL) and grown in supplemented Dulbecco’s Minimum Essential Medium (DMEM). After 24 h, the medium was replaced with 200 μL of fresh culture medium containing the CDs at range of concentration from 1 to 0.05 mg mL⁻¹. After 48 h, MTS assay was performed replacing the dispersion of CDs in DMEM with 100 μL of fresh medium and 20 μL of MTS solution. After 2 h of incubation at 37 °C and 5% of CO₂, the absorbance at 492 nm was measured using a microplate reader (Eppendorf PlateReader AF2200, Hamburg, Germany). Untreated cells was used as negative control. In another experimental set was evaluated the NIR-triggered photothermal ablation on MDA-MB-231 of the selected fractions of CDs_18.5bar, CDs_13bar and CDs_8bar. Cells were seeded in a 96 multiwell as described above. After 48 h of incubation with the dispersions of CDs in DMEM (1–0.05 mg mL⁻¹), each well was irradiated with an 810 nm laser for 300 s with power fitted at 5 W cm⁻². The hyperthermic effect was reported as percentage reduction of the control cells. All culture experiments were performed in triplicate.

Visual autoaggregation behavior of the 30 isolates was performed according to Simoes et al. [27 (link)] and Cisar et al. [6 ] with certain modifications. Bacteria were cultivated for 24 h at 28 °C in LB medium with the following composition g L⁻¹: 10 tryptone (Sigma-Aldrich), 10 NaCl (Sigma-Aldrich), and 5 yeast extract (BioMérieux). The cells were harvested at stationary phase by centrifugation at 5000×g for 15 min, washed twice, and resuspended in phosphate-buffered saline (PBS, pH 7.3) and adjusted to OD at a wavelength of 600 nm to approximately 1.0 (10⁸ CFU ml⁻¹). The bacterial suspensions (2.5 ml) were mixed by vortexing by 10 s, then transferred onto 24-well plate (Falcon), and allowed to settle. Degree of autoaggregation was determined after 0, 2, and 24 h of incubation at 28 °C. After the incubation period, a 0.2 ml of upper portion of the suspensions was transferred onto 96-well plate and OD₆₀₀ was measured (Plate Reader AF2200, Eppendorf). The autoaggregation percentage was expressed as (1 − A_t)/A₀ × 100, where A_t means the absorbance at time t = 2 and 24 h and A₀ the absorbance at time t = 0. All experiments were performed in triplicate.

Hydrogen peroxide was quantified using the Amplex Red hydrogen peroxide/peroxidase assay kit (Invitrogen catalog no. A22188) as previously demonstrated but with minor modifications (30 (link)). Three female adult D. melanogaster flies were collected and homogenized in 300 μl of PBS with three 2-mm glass beads beating for 10 s at 4,200 rpm. For Canton-S flies, heads were removed because of the intense red eye pigment. Samples were centrifuged at 12,000 × g for 3 min (room temperature), and 50 μl of supernatant was used for the assay following the manufacturer’s protocol with spectrophotometry quantification at 560 nm or excitation/emission of 535/595 nm using a BioTek Eon microplate reader or Eppendorf PlateReader AF2200, respectively. Hydrogen peroxide concentrations were normalized to total protein and plotted as relative H₂O₂ to the vehicle. Total protein was quantified using a bicinchoninic acid (BCA) protein assay kit (Invitrogen catalog no. 23227) following the manufacturer’s microplate protocol. Protein was measured from samples that were obtained from the H₂O₂ determination protocol and used to normalize H₂O₂ quantification. Samples were centrifuged at 12,000 × g for 3 min (room temperature), and 25 μl was used for quantification as per the manufacturer’s microplate protocol using a BioTek Eon microplate reader at 562 nm.

OCs were prepared using the following procedure in all experiments: RAW264 cells were seeded first in a 96-well plate at a density of 3.1×10³ cells/cm² and cultured in αMEM supplemented with 10% FBS and penicillin-streptomycin-amphotericin B suspension. Twenty-four hours after incubation, a receptor activator of nuclear factor kappa-B ligand (RANKL; Oriental Yeast Co., Ltd., Tokyo, Japan) was added to the cultured medium at a concentration of 200 ng/mL and further incubated for 4 days.
OBs were seeded in a 96-well plate at a density of 3.1 × 10⁴ cells/cm². After 24 h, CNMs were added to the cultures and incubated for 24 or 48 h. The cell viability was analyzed using alamarBlue® reagent (Bio-Rad, Hercules, CA, USA) according to the manufacturer’s instructions. Briefly, the alamarBlue® reagent was diluted in the medium (1:10) and added to the cultures. After 1 h of incubation, fluorescence intensity was measured using a PlateReader AF2200 (Eppendorf, Hamburg, Germany) with the excitation/emission wavelengths set at 535/590 nm. The group to which only the dispersant was added was designated as the control, and cell viability was calculated with the control set as 100%.

Two slices of formalin-fixed, paraffin-embedded (FFPE) tissue samples (about 10 mg of tissue) were used for total genomic DNA extraction. Samples were pre-treated at 55 °C for a minimum of 1 h with the dissolving buffer and Proteinase K (20 mg/ml) according to the manufacturer’s instructions (BiOstic® FFPE Tissue DNA Isolation Kit Mobio, Carlsbad, CA, USA). DNA was used to study the microbial diversity by pyrosequencing of the amplified V1–V3 region of the 16S rRNA gene, recurring to the primers Gray28F (5’-TTTGATCNTGGCTCAG) and Gray519r (5’-GTNTTACNGCGGCKGCTG) that amplify a fragment of 520 bp, following PCR conditions previously reported [17 (link)]. PCR products were purified twice with Agencourt AMPure purification kit (Beckman Coulter, Milan, Italy), and quantified using the PlateReader AF2200 (Eppendorf, Hamburg, Germany) with PicoGreen assay and an equimolar pool was obtained prior to further processing. Due to poor DNA quality, PG_8 sample was excluded. The amplicon pool was used for pyrosequencing on a GS Junior platform (454 Life Sciences, Roche, Monza, Italy) according to the manufacturer’s instructions by using Titanium chemistry.

The MC3T3-E1 cells were seeded in 96-well plates at a density of 1.0 × 10⁵ cells/mL and cultured in αMEM containing 10% FBS and 1% penicillin-streptomycin. After 24 h of culture, NFPs, 100 µg/mL of ascorbic acid (Nacalai Tesque), and 5 mM of β-glycerophosphate (Calbiochem, LaJolla, CA, USA) were added to the culture for an additional 24 or 48 h. Cell proliferation was assayed using the alamarBlue^® reagent (Bio-Rad, Hercules, CA, USA). After 45 min of incubation with the diluted alamarBlue^® reagent (1:10 in DPBS), the fluorescence intensity was measured using PlateReader AF2200 (Eppendorf, Hamburg, Germany) at excitation/emission wavelengths of 535/590 nm [26 (link)]. The control group treated only with the dispersant was designated as 100%, and the cell proliferation was calculated relative to this control.