Trypan blue solution

Zirconyl chloride (ZrOCl₂, 8H₂O), phosphoric acid and Methylene blue were obtained from Merck. DMEM medium (Dulbecco’s Modified Eagle Medium) was purchased from Invitrogen. MTT assay reagent, (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide), was supplied by Sigma-Aldrich. Trypan blue solution (0.4% w/v) and dimethyl sulfoxide (DMSO) were achieved from Merck Company. Fetal bovine serum (FBS) and antibiotics were purchased from Gibco (Gibco BRL). All chemicals were of analytical grade. Double distilled deionized water was used for all experiments and solutions. All pH measurements were made at 25 °C using Bel PHS3-BW (BEL ENGINEERING, Italy). The UV-Vis absorption spectra were recorded using Cary 60 UV/Vis spectrophotometer, equipped with quartz cells. Red light emitting LED (660 nm; power density: 30 mW cm⁻²) was used as light source for photodynamic experiments.

A total of 8 × 10⁴ cells were plated in the upper chamber of cell culture inserts (Falcon, 353495) coated with (for the invasion assay) or without (for the migration assay) Matrigel (Corning, Corning, NY, USA) and incubated at 37 °C in 5% CO₂ for 24 h. The migrated cells retained on the bottom surface of the membrane were fixed and stained with trypan blue solution (Merck, Branchburg, NJ, USA). The nonmigrated cells were scraped from the upper surface of the membrane using a cotton swab. The cells were photographed and quantified using ImageJ software.

RT112 and J82 cell lines from ATCC were available at the Institute of Food Science, Avellino, Italy; Roswell Park Medium Institute (RPMI) and Minimum Essential Medium Eagle (EMEM) medium, fetal bovine serum (FBS), L-glutamine 200 mM, penicillin 5000 IU/mL/streptomycin 5000 μg/mL, and PBS (phosphate buffer saline) were purchased from Lonza (Euroclone SPA, Pero, Italy); Crystal Violet, trypan blue solution (0.4% v/v), RNase A, propidium iodide, quercetin, rutin, myricetin, gallic acid, kaempferol and dimethylsulfoxide (DMSO) were from Merck Life Science (Milano, Italy). Glutathione (GSH); phtaldialdehyde, 2′,7′-dichlorofluorescein diacetate (DCFH-DA), N-acetyl-L-cysteine (NAC), L-Buthionine-sulfoximine (BSO) were from Carlo Erba (Milan, Italy). Reagents for Folin–Ciocalteau were purchased from Merck Life Science (Milan, Italy). Methanol, water (HPLC-grade), and trifluoroacetic acid were purchased from Merck-Schuchardt (Hohenbrunn, Germany). Hydroxytyrosol, tyrosol, oleocanthal, oleuropein aglycone, and ligstroside aglycone analytical standards were purchased from Merck-Schuchardt (Hohenbrunn, Germany).