Dulbecco modified eagle medium (dmem)

The human MCF-7 and MDA-MB-231 cells were obtained from ATCC (Manassas, VA, USA) and cultured in RPMI-1640 with 10% FBS (Sigma, St. Louis, MO). MCF-10A and HMLE cells were cultured in DMEM/F12K supplemented with growth factor hEGF, hydrocortisone, insulin and 100 units of penicillin/ml and 100 mg of streptomycin/ml. HEK-293T cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM) (Cambrex) supplemented with 10% FBS. All media contained 2 mM glutamine, 100 units of penicillin/ml and 100 mg of streptomycin/ml. Cells were incubated at 37°C and supplemented with 5% CO₂ in a humidified chamber. For experiments, cells were grown in medium with exosome-free serum. Therefore, the serum added to the cell culture medium was depleted of exosomes by ultracentrifugation at 120,000 × g overnight (16 h) at 4°C, followed by passing it through 0.2 micron filter prior to use.

LR7 cells were maintained in Dulbecco’s Modified Eagle Medium (DMEM; Cambrex Bioscience, Walkersville, MD) supplemented with 10% fetal calf serum (Bodinco Alkmaar, The Netherlands), 100 IU of penicillin/ml and 100 µg/ml of streptomycin (both from Life Technologies, Rochester, NY). Wild type MHV-A59 was propagated in LR7 cells in DMEM.

Human adrenergic neuroblastoma, cell line SH-SY5Y (Sigma-Aldrich), were supplemented with 10% (v/v) inactivated Fetal Calf Serum (FCS) (Biowest, Nuaillé, France), 2 mM L-glutamine (Biowest), 100 IU/ml penicillin and streptomycin in 25 cm² flasks at the cell density of 10⁶ cells/ml in Dulbecco's Modified Eagles Medium (DMEM, Cambrex BioScience, Verviers, Belgium). They were then cultured in a humidified atmosphere of 5% CO₂ at 37°C. The medium was replaced every 2 days and the cell cultures were split twice a week. Cells at about 80% of confluence were trypsinized (trypsin-EDTA, Biowest), washed and scored for viability. Culture medium was replaced with fresh one after overnight recovery and cells were categorized into four groups, each consisting of about 20×10⁶ cells. The four groups of cells marked as: i) untreated, negative control (−SMF/−cisPt), ii) treated with 0.1, 0.5, or 1 µM cisPt (−SMF/+cisPt), iii) exposed to SMF (+SMF/−cisPt), and iv) treated with cisPt and exposed to SMF (+SMF/+cisPt). Different treatment regimens (±cisPt and/or ±SMF) were carried out for 2, 4 or 24 h, continuously. Biochemical and morphological investigations were done after each treatment.

All reagents were used without further purification. Chitosan (CH) medium molecular weight and glacial acetic acid were purchased from Sigma–Aldrich (Milano, Italy). Aliette by Bayer was applicated as commercial formulation of fosetyl-Al (Fos). For cells culture and experiments, the following reagents are used: Dulbecco’s Minimum Essential Medium (DMEM) (Cambrex, Verviers, Belgium), fetal calf serum (FCS), glutamine (Cambrex, Verviers, Belgium), penicillin and streptomycin solution (Cambrex, Verviers, Belgium), Spurr resin (TAAB, Berks, UK), tolulidine blue (Sigma-Aldrich, Milano, Italy), cacodilate buffer and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide salt (MTT) (Sigma-Aldrich, Milano, Italy). For antibacterial test: Gentra Puregene Yeast/Bact. Kit was purchased from Qiagen (PL Venlo, The Netherlands), SYBR select master mix from Applied Biosystem (Foster City, CA, USA).

Human monocytes were obtained from healthy donor buffy coats by two-step gradient centrifugation using Ficoll (Biochrom) and Percoll (Amersham) followed by incubation of purified cells in RPMI 1640 (Lonza) without serum for 10 min at 37°C with 5% CO₂. Adherent monocytes were washed twice with PBS and then cultured with RPMI medium supplemented with 10% FBS and L-glutamine as fully described below. The purity of the monocytes cultures was tested by CD14 staining and flow cytometry analysis, with an average of 90% CD14⁺ cells.
Monocytes and THP-1 cells (ATCC) were grown in RPMI supplemented with 10% heat-inactivated fetal bovine serum (FBS; Lonza), 100 U/mL penicillin/streptomycin (Lonza), and 25 mM L-glutamine (Lonza) at 37°C with 5% CO₂. HEK-293T cells (ATCC) were grown in D-MEM (Cambrex) supplemented with 10% FBS, 100 U/mL penicillin/streptomycin, and 25 mM L-glutamine at 37°C with 5% CO₂.

Acridine orange, 2,3-diaminonaphthalene (DAN), 5,5′,6,6′-tetrachloro-1,1′,3,3′-tetraethyl-benzimidazol-carbocyanine iodide (JC-1), 2′,7′-dichlorodihydrofluorescein diacetate (DCFH₂-DA), and Autophagy Assay kit were obtained from Sigma Aldrich cat. no.:MAK138 (St. Louis, Missouri, USA). Human MAP1LC3A (Microtubule-associated proteins1A/1B light chain 3A) ELISA Kit was obtained from Fine-test (Wuhan, Hubei, China). The Fluo-4 NW Calcium Assay Kit was obtained from Molecular Probes (Eugene, USA). The Sphingomyelinase Assay kit was purchased from Abcam (Cambridge, UK). Culture media (DMEM, RPMI 1640) and fetal bovine serum (FBS) were obtained from Cambrex (Basel, Switzerland); trypsin-EDTA, penicillin and streptomycin were acquired from Sigma Aldrich (St. Louis, Missouri, USA).