Arpe 19 cell line

Compritol 888 ATO was obtained from Gattefossè (Milan, Italy), oil Miglyol 812 was provided by Eigenmann & Veronelli S.p.A. (Milan, Italy) and Lutrol F68 was purchased from BASF ChemTrade GmbH (Burgbernheim, Germany). Mangiferin, Phosphate Buffered Saline (PBS commercial 10×) and all solvents were purchased from Merck (Milan, Italy). Ultrapure water (resistivity > 18.2 MΩ·cm) was obtained by reverse osmosis (Milli-Q, Millipore Iberica, Madrid, Spain). The ARPE 19 cell line was purchased from ATCC, and bromide 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium (MTT), dimethyl sulfoxide (DMSO), sulfhydryl reagent 5,5′-dithio-bis (2-nitrobenzoic acid) (DTNB) and 2′,7′- dichlorofluorescein diacetate (DCFH-DA) were purchased from Sigma–Merk Life Science, Milan, IT.

The cytotoxicity effects of NADES and ACZ/NADES mixtures were evaluated using the ARPE-19 cell line (ATCC), which is an immortalized human spontaneously arising retinal pigment epithelia cell line.
ARPE-19 cells were incubated for 24 h in a 96-well plate at a density of 1.0 × 10⁴ cells/well and then incubated for another 48 h until confluency was reached (ca. 70%). Afterwards, selected NADES and NADES/ACZ mixtures were added at 5% (w/v) and 10% (w/v) concentrations and incubated for 24 h at 37 °C and 5% CO₂. Samples were prepared by dissolving the samples in culture media.
Control cells were incubated with complete media. To evaluate cell viability, CellTiter 96^® AQ_ueous One Solution Cell Proliferation Assay (Promega), based on MTS (3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetra-zolium), was carried out.

A human ARPE-19 cell line was purchased from ATCC and was cultured in DMEM-F12 medium supplemented with 10% fetal bovine serum, 0.348% sodium bicarbonate, 2 mM L-glutamine, 100 U/mL penicillin, and 100 μg/mL streptomycin. Cell cultures were maintained at 37°C in a humidified atmosphere of 95% air and 5% CO₂. Medium was changed every two days. ARPE-19 cells were used within 10 generations.

Avastin^® Roche (Basilea, Switzerland) was kindly purchased by Molinette Central Hospital (Turin, Italy). Deionized water was obtained by a Milli-Q system (Millipore, Bedford, MO, USA). Tween^® 20 (polysorbate 20), Tween^® 80 (polysorbate 80), hydroxyethyl cellulose, sodium phosphate monobasic and sodium phosphate dibasic were purchased from ACEF (Fiorenzuola d’Arda, Italy); trehalose dihydrate, cefuroxime, decyl polyglucoside, ethyl oleate, Pluronic^® F127, dioctyl sodium sulfosuccinate (AOT) from Merck (Darmstadt, Germany); Epikuron^® 200 (phosphatidylcholine 92%) from Cargill (Minneapolis, MN, USA); hyaluronic acid, sodium sulfate and sodium chloride from Alfa-Aesar (Ward Hill, MA, USA); Labrasol^® from Gattefossè (Saint-Priest, France).
Sulforhodamine B (SRB), dimethyl sulfoxide (DMSO), trichloroacetic acid, fetal calf serum and antibiotics for cell cultures were all purchase from Sigma-Aldrich (St. Louis, MO, USA). The retinal pigment epithelial ARPE-19 cell line (ATCC-CRL-2302) and the DMEM:F12 medium was purchase from ATCC^® (Manassas, VA, USA).

The human ARPE-19 cell line was purchased from the ATCC (Manassas, VA). The cells were routinely grown in DMEM/F12 medium (WelGENE, Korea) supplemented with 10% heat-inactivated fetal bovine serum (FBS; Gibco BRL, Grand Island, NY), 2 mM glutamine, 100 U/mL penicillin and 100 μg/mL streptomycin. The cells were cultured at 37°C in 5% CO₂, and passaged every 3–4 days. The viability of the ARPE-19 cells was assessed by staining with trypan blue dye.
The RH tachyzoites of T. gondii expressing GFP or RFP were maintained by in vitro ARPE-19 cells at 37°C in 5% CO₂. After spontaneous host cell rupture, parasites and cellular debris were pelleted by centrifugation and washed with cold PBS. The final pellet was resuspended and passed through a 26-gauge needle and finally filtered through 5.0-μm pore sized filter (Millipore, Billerica, MA) to obtain a tachyzoite suspension free of host cell debris.