Edta solution

Normal and cancer gingival cells (3 × 10⁵ cells/well) were seeded in 6-well plates and cultured for 24 h. Cells were then exposed or not to various concentrations of anethole for 24 h, then sued to assess the cell growth by means of MTT colorimetric assay (Sigma-Aldrich) as described by Semlali et al.^{20 (link)} Briefly, each culture was supplemented with 10% of MTT (5 mg/mL) and incubated for 3 h at 37 °C. Stained cells were then lysed using 1 mL of isopropanol-HCl 0.05 M solution with agitation for 15 min. Then, 4 × 200 µL of lysis solution were transferred to a 96-well plate and the absorbance was measured at the wavelength of 550 nm using an iMark microplate reader (Bio-Rad, Mississauga, ON, Canada). Cell proliferation levels were determined by means of the following formula: % of cell viability = [(OD _550 nm (treated cells) − OD (Blank))/(OD (control cell) − OD (Blank))] × 100, n = 7. With another set of experiments, anethole treated and non-treated gingival cells were detached by means of 0.05% trypsin: 0.1% EDTA solution (Sigma-Aldrich, Oakville, ON, Canada) and resuspended in fresh culture medium. The level of living cells in each culture was determined by means of the trypan blue exclusion assay, as described previously^{21 (link)}.

Fresh peripheral venous blood was collected from healthy volunteers via the Regional Blood Donation Center (Krakow, Poland), which complies with the requisite confidentiality assurances for human participants. Thus, the current work adheres to appropriate exclusions from human subjects’ approval. The blood sample was diluted in 5 mM EDTA solution (Sigma) and centrifuged at 300 g for 15 min to remove the plasma layer. The remaining suspension was diluted with Mg²⁺- and Ca²⁺-free phosphate-buffered saline (PBS) (Biowest, Nuaille, France) and overlaid on the lymphocyte separation medium (Biowest). After centrifugation at 300 g for 30 min, the pellet-containing neutrophils and red blood cells (RBCs) were subjected to 1% polyvinyl alcohol (Sigma) sedimentation to separate the RBCs from the neutrophils. Residual RBCs were lysed by Red Blood Lysis Buffer (Roche, Penzberg, Germany) and then washed with PBS. The number of cells was determined using a Bürker chamber. The neutrophils’ purity was assessed routinely by forward- and side-scatter flow cytometric analyses. This neutrophil isolation procedure yields a >95% pure population of the cells.

Presence of DFX in the MSC secretomes was evaluated by High Performance Liquid Chromatography (HPLC) based on a modified method reported by DeFrancia et al. [29 ]. A VWR Hitachi LaChromElite chromatographic system equipped with autosampler, column oven adjusted to 25°C, and a UV-DAD detector adjusted to 430nm was used. In order to prevent metal interference, the entire system was previously purged with a 20mM EDTA solution (Sigma-Aldrich). Deferoxamine was evaluated in samples of the MSC secretome before the washing steps and after the first and second washing steps. For this, 50μl of each sample were first complexed with 50μl of a 3mM ferric chloride solution (Sigma-Aldrich). Samples were incubated for 5 minutes to obtain the ferroxamine form and 5μl of this solution were injected into the chromatographic system with a mobile phase consisting of 15% methanol in an aqueous solution containing 0,5% triethylamine adjusted to pH 8.3. Samples were separated on a Kromasil® C18 column (4,6 x 100 mm) with 3.5μm particle size through a flow rate of 0.8ml/min. Signals were quantified using a six point DFX standard calibration curve from 10μM to 2000μM (r² 0.998).

On the first day of hospitalization, the pregnant women were interviewed, submitted to oral cavity examination, and non-stimulated saliva collection. Saliva of neonates was collected shortly after birth and prior to any contact with the mother, including the first breastfeeding, to avoid contamination with non-salivary components. Saliva samples were collected using sterile polypropylene transfer pipettes. A 250mM EDTA solution, pH 5.2 (Sigma, St Louis, MO, USA) was added to each sample prior to transport on ice to the laboratory, where they were stored at - 80ºC until analysis.
Samples of colostrum were collected by manual expression into sterile polypropylene Falcon tubes, on the first day of lactation. After collection, the samples were transported on ice to the laboratory, centrifuged at 1,300g, for 5 minutes, and stored at -80ºC until the laboratorial procedures.

Flocculation was determined using the microflocculation method (21 (link)). Total cell number was calculated by extrapolation of the absorbance values obtained in each standard cell curve, and the number of flocculent cells was calculated by subtracting the number of cells that remained in the suspension after stopping the agitation, according to Ogata (28 (link)). As a negative flocculent control, the assay was conducted using deionized water instead of EDTA solution (Sigma-Aldrich, Merck). Experiments were performed in triplicate.

The in vitro toxicological study was conducted in a phagocytic cell line murine, RAW 264.7 (mouse leukaemic monocyte macrophage cell line, cultures from ECACC (European Collection of Authenticated Cell Cultures) supplied by Sigma 85062803).
The macrophage cell line was grown in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum, 2 mM L-glutamine, penicillin (100 UI/ml) and streptomycin (100 μg/ml) and maintained at 37°C in a humidified atmosphere (95%) under 5% CO₂. Cells were seeded at approximately 10,000 cells/cm² and passaged every 3±4 days as necessary; they were finally removed from the plates with 0.05% trypsin-0.02% EDTA solution (all cell material was purchased from Sigma-Aldrich).