Eagle s minimum essential medium

Human recombinant insulin, acridine orange, ethidium bromide, all-trans retinoic acid (RA), dichlorofluorescein diacetate (DCFDH), 5,5′-dithiobis-(2-nitrobenzoic acid) [DTNB], Ham's F-12, Hoechst 33342, Eagle's minimum essential medium (MEM), and trypan blue were purchased from Sigma-Aldrich, USA. 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide, disodium hydrogen phosphate dihydrate, monosodium glutamate monohydrate, and fetal bovine serum were procured from HiMedia Laboratories, India.

The chemicals tetraethyl orthosilicate (TEOS), n-cetyltrimethylammonium bromide (CTAB), sodium hydroxide (NaOH), safranin O, glutathione (GSH), (3-mercaptopropyl) trimethoxysilane (MPTMS), 2,2′-dipyridyl disulfide, peroxidase from horseradish (HRP), H₂O₂ 30%, β-glycerophosphate, and poly(ethylene glycol) methyl ether thiol (Mn 800) were provided by Aldrich. Doxorubicin hydrochloride (DOX) was supplied by Sequoia Research Products. For cell culture studies, U271 malignant glioma cells, Eagle’s minimum essential medium (MEM), penicillin–streptomycin, phosphate-buffered saline (PBS), fetal bovine serum (FBS), glutamine, trypsin, and cell proliferation reagent (WST-1) were obtained from Sigma-Aldrich. Tyramine-derivatized hyaluronic acid was purchased from Contipro A.S. Ultra-pure chitosan (UP CL214, Protasan) was supplied from Pronova Biomedical (Norway). The analytical-grade solvents were provided from Scharlab (Barcelona, Spain). All reagents were used as received.

Eagle’s minimum essential medium (MEM) was purchased from Sigma (Milan, Italy); trypsin-EDTA, penicillin, streptomycin, sodium pyruvate, non-essential amino acid solution, fetal calf serum (FCS), plates and Petri dishes were purchased from EuroClone (Pero, Milan, Italy). 3,3’-Dioctadecyloxacarbocyanine perchlorate (DiO) was purchased from Sigma (Milan, Italy). Berberine and sucrosomial^® berberine were provided by PharmaNutra S.p.A. and were prepared by dissolving 10 mg of extracts in 1 mL of water for the in vitro studies; otherwise, a 15 mg/mL suspension was prepared in saline solution (0.9% NaCl).

Paclitaxel (PTX), Mw = 853.906 g/mol, was obtained from LC laboratories (Woburn, MA, USA). Poly(d,l-lactide-co-glycolide) (PLGA) 50:50, Mw = 7000–17,000 Da (PLGA-7K), 24,000–38,000 Da (PLGA-24K) and 38,000–54,000 Da (PLGA-38K); Kolliphor^® P188 (Kol) (poly(ethylene glycol)-block-poly(propylene glycol)-block-poly(ethylene glycol); N-methyl pyrrolidone (NMP), acetonitrile HPLC grade 99% purity, Eagle’s minimum essential medium (MEM), fetal bovine serum (FBS) and curcumin (CUR, Mw = 368.38 g/mol), were purchased from Sigma-Aldrich Co. (St. Louis, MI, USA).

Cell lines were obtained from the Coriell Institute for Medical Research (Camden, NJ, USA). The Coriell Cell Repository maintains the consent and privacy of the donor samples. All cell lines and culture protocols in the present study were carried out under institutional review board guidelines at University of Lleida and the IRBLleida research center. Two human fibroblast cell lines from patients with SMA (GM03813, SMAII; and GM09677, SMAI) and one unaffected control (GM03814, Control) were purchased and cultured following manufacturer instructions. Cells were maintained in Eagle's Minimum Essential Medium (MEM) (Sigma) supplemented with non-inactivated fetal bovine serum (FBS; Gibco) (15% v/v), 0.5 M of L-Glutamine (Gibco), non-essential amino acids (Gibco) (1% v/v), and 20 μg/ml Penicillin–Streptomycin (Gibco). Cells were subcultured every 3–4 days. For western blot analysis, cells were plated at 3000–4000 cells/cm² in 35 mm tissue-culture dishes and maintained in supplemented MEM. Two days later, total cell lysates were collected and submitted to western blot analysis. For immunofluorescence experiments, 5,000 cells/well were plated on 4-well dishes with collagen-coated 1 cm² glass coverslips, maintained in the MEM for 24 h, then fixed in 4% paraformaldehyde in PBS.

Eagle’s minimum essential medium (MEM) was bought from Sigma, trypsin-EDTA, penicillin, streptomycin, sodium pyruvate, non-essential amino acid solution, fetal bovine serum (FBS), plates and Petri dishes were bought from EuroClone (Milan, Italy). YVVNPDNNEN peptide corresponds to position 294–303 of the α subunit of β-conglycinin (UNIProtKB P0DO16), whereas YVVNPDNDEN peptide corresponds to positions 310–319 of the α′ subunit of β-conglycinin (UNIProtKB P11827). These peptides were certified >95% purity by HPLC (PRIMM; Milan, Italy). Simvastatin was bought from Sigma-Aldrich (Milan, Italy).