Caco 2 cells

Caco-2 cells were purchased from European Collection of Cell Cultures (ECACC, Public Health England Porton Down, Salisbury, UK). Cell medium, substances and reagents for cell cultures were all purchased from Sigma-Aldrich (St. Louis, MO, USA), unless stated otherwise. Instruments, reagents and materials for Western blot analysis were obtained from Bio-Rad Laboratories (Milan, Italy). S100B protein was from Sigma-Aldrich (St. Louis, MO, USA) and mouse monoclonal S100B antibody (S100BmAb) was purchased from AbCam (Cambridge, UK). Mouse anti-total Akt, mouse monoclonal anti p-ERK/ERK was from Santacruz biotechnology (DBA, Milan, Italy) rabbit monoclonal anti-phospho-Akt (Ser⁴⁷³), rabbit polyclonal anti-phospho-mTOR (pSer²⁴⁴⁸), rabbit monoclonal anti VEGF-R1 and VEGF R-2 were purchased from Cell Technology (Euroclone, Pero, Milan, Italy). Mouse monoclonal anti RAGE antibody was from AbCam (Cambridge, UK). Rabbit polyclonal anti-total mTOR was from Abcam (Cambridge, UK); mouse monoclonal anti-HIF1α was purchased from Sigma Aldrich (Milan, Italy); anti-β-actin was from Santa Cruz Biotechnology (Santa Cruz, California, USA). SB203580 was from InvivoGen (Aurogene, Rome, Italy) and polyclonal rabbit anti-mouse IgG from Dako (Glostrup, Denmark).

All the reagents and solvents were of reagent grade, used without any further purification. Ascorbic acid (AA), ascorbate oxidase (AOx, EC 1.10.3.3), 2,2-diphenyl-1-picrylhydrazyl (DPPH) free radical, Rutin, 2,2’-Azino-bis(3-ethylbenzothiazoline-6-sulfonic acid, diammonium salt (ABTS), 2′,7′- dichlorodihydrofluorescein diacetate (H₂DCFDA), tert-butyl hydroperoxide (TBH), trichloroacetic acid (TCA), 2-thiobarbituric acid (TBA), methanol, 1,1,3,3-tetraethoxypropane (TEP), thiazolyl blue tetrazolium bromide (MTT), isopropanol, streptomycin, penicillin, ampicillin, oxacillin, cloxacillin, rifampicin, oxytetracycline, and L-glutamine were purchased from Sigma-Aldrich (Milan, Italy). The phosphate-buffered saline (PBS) solution was made using NaCl (137 mM), NaOH (2.7 mM), Na₂HPO₄ (8.1 mM), and KH₂PO₄ (1.47 mM) from Sigma and then adjusted to pH 7.4. Dulbecco’s modified Eagle’s medium (DMEM), RPMI 1640 and fetal bovine serum (FBS) were purchased from Euroclone S.p.A. (Pero, Milan, Italy).
CaCo-2 cells were obtained from the European Collection of Cell Cultures (ECACC, Salisbury, UK). SW48 were purchased from the American Type Culture Collection (ATCC, LGC Standards S.r.l., Milan, Italy).

Rat pheochromocytoma-derived cell line (ATCCCRL-1721) PC12 cells (passage 12–25) were cultured in atmosphere of 5% CO₂/95% humidified air at 37 °C in 60 mm plastic culture plates in Dulbecco’s modified Eagle’s medium (DMEM/F12) with 10% Horse Serum (HS), 5% Fetal Bovine Serum (FBS) and 1% of penicillin/streptomycin. CACO-2 cells (European Collection of Cell Cultures (ECACC UK) (passage 20–40) were maintained in tissue culture flask T75 in Dulbecco’s modified Eagle’s medium (DMEM) with 10% FBS, 1% nonessential amino acids, 1% L-glutamine and 1% penicillin–streptomycin solution and maintained at 37 °C and 5% CO₂. The experiments were performed in the cell lines exposed to the different beta-cyclodextrin monomers and polymers (HP, QA, QAPS, HA, HAPS and PS) at different concentrations and times of exposure.

Dulbecco’s Modified Eagle’s Medium (DMEM) with 4.5 g/L glucose, L-glutamine, sodium pyruvate and sodium bicarbonate, Hank’s Balanced Salt Solution (HBSS) modified with sodium bicarbonate (without phenol red), non-essential amino acids (NEAA), antibiotic/antimycotic solution (penicillin, streptomycin, and amphotericin) and foetal bovine serum (FBS, non-USA origin), Curcumin (CUR), Triton X-100 and QuantiPro^TM BCA Assay Kit were purchased from Sigma-Aldrich (Poole, UK). Caco-2 cells were purchased from the European Collection of Cell Cultures (ECACC) and used between passages p66–p74. MTS reagent, namely 3-(4,5-dimethylthiazol-2-yl)-5(3-carboxymethonyphenol)-2-(4-sulfophenyl)-2H-tetrazolium (commercially known as CellTiter 96 AQueous One Solution Assay) was purchased from Promega (Madison, WI, USA). Exosome isolation kit from other body fluids and exosome isolation kit form cell culture were purchased from Thermo Fisher Scientific (Loughborough, UK), ExoGlow™-Protein EV Labeling Kit (Red) and exosome-depleted FBS were purchased from System Biosciences (Palo Alto, CA, USA). Transwell^® permeable cell culture inserts (polycarbonate filter, 1.1 cm² diameter, 0.4 μm pores) were obtained from Corning (Corning, NY, USA). PD-10 columns, Sephadex G-25 were obtained from GE Healthcare (Chicago, IL, USA).

Caco-2 cells were supplied by the European Collection of Cell Cultures (ECACC, Salisbury, UK). Cells were cultured in minimum essential media (MEM) containing 20% fetal bovine serum (FBS), 1% sodium pyruvate, sodium bicarbonate 2.2 g/L, gentamicin 5 mg/L, streptomycin 10 mg/L, penicillin G 6 mg/L. Cells were maintained in a 37 °C, 5% CO₂ humidified incubator until approximately 75% confluent. All the following in vitro experiments were carried out in FBS-free media. Cells were incubated with 3 series of test compounds at 10 µM for 30 min. The dose of 10 µM was chosen in accordance with our previous study were toxicological studies were carried out [17 (link)]. Cytokines TNF-α and IL-1β 10 ng/mL were then added and the cells were incubated for 24 h. For NO-donor experiments, solutions of SNAP or DETA-NONOate were prepared daily when needed and incubated (in the concentration range 10 to 500 µM). For co-incubation experiments with ODQ, this was added with the nitrate-barbiturate hybrids to give a concentration in the serum-free media of 10 µM as with the compounds. In experiments where 8-Br-cGMP was used, this was added with the ODQ and compounds to give a final concentration of 10 µM in the serum-free media.

Caco-2 cells were purchased from the European collection of cell cultures (ECACC, health protection agency, UK). Cells were cultured in 24 – well plates at a density of 25.000 cells/well with minimum essential medium (MEM) supplemented with 10% fetal bovine serum (FBS) and 1% penicillin-streptomycin at 37 °C and 5% CO₂ atmosphere (95% relative humidity). The old MEM was changed on alternate days until a cell monolayer was observed. Prior to the experiment, cells were washed twice with 500 μl of glucose – HEPES buffer (268 mM glucose and 25 mM HEPES at pH 7.4). The control cells were incubated with 500 μl of glucose-HEPES buffer containing 1% (v/v) phosphatase inhibitor cocktail for 1 h prior to experiment. Afterwards, cells were washed again and incubated with 500 μl of 0.01% (m/v) Cs-PP NP suspension in glucose-HEPES buffer. Aliquots of 50 μl were transferred to 96 well plates at set time intervals (0, 30, 60, 90, 120, 150 and 180 min). As a control the experiment was performed under equivalent conditions with samples containing 1% (v/v) phosphatase inhibitor cocktail. Released monophosphate was evaluated via MLG assay as described in section 2.5.

Colorectal-carcinoma-derived epithelial Caco-2 cells (European Collection of Cell Cultures) and airway epithelial Calu-3 cells (American Type Culture Collection; ATCC) were cultured in DMEM supplemented with 10% of FBS and 1% of an antibiotic–antimycotic mixture. Erythrocytes were collected from mice blood. The cytotoxicity of the N-benzyl quaternary ammonium surfactants derived from leucine in comparison to the commercial BAC was studied by performing two cytotoxicity assays (MTS and LDH as indicators of metabolic activity and membrane integrity, respectively) and by a haemolytic assay. The assays were conducted as reported in our previous publications [5 (link),14 (link)]. EC₅₀ values from the MTS assay (concentration of surfactant that causes 50% cell death) and the LDH assay (concentration of surfactants that causes 50% LDH release from cells), as well as HC₅₀ values from the haemolytic assay (concentration of surfactants that causes 50% haemoglobin release from erythrocytes) were calculated using a nonlinear regression model (Prism version 5.0, GraphPad Inc., San Diego, CA, USA) as follows: $Y=\mathrm{BOTTOM}+\frac{\mathrm{Top}-\mathrm{Bottom}}{1+{10}^{\left(\mathrm{LogEC}50-\mathrm{x}\right)\mathrm{Hill}\mathrm{Slope}}}$
where Top and Bottom are plateaux in the units of the Y axis.