Ethylene diamine tetraacetic acid (edta)

Primary skin fibroblast cell lines were isolated from skin biopsies as follows. Skin specimens were washed with 70% ethanol and physiological solution and incubated with 2 mg/mL Dispase II (Merck KGaA, Darmstadt, Germany) overnight at 4 °C. Then, the dermis was separated using sterile tweezers, cut into 2–4 mm pieces and plated on a 6-well microplate. A squared sterile glass was put above and 1 mL of high glucose Dulbecco’s modified Eagle’s medium, DMEM (Euroclone, Milan, Italy) supplemented with 20% fetal bovine serum, FBS (Euroclone, Milan, Italy) was added to each well. After 3 weeks, dermis and glasses were removed and fibroblasts were detached with 0.25% Trypsin and 0.02% EDTA (Euroclone, Milan, Italy) and plated in 25 cm² flasks. Cells were then cultured in DMEM supplemented with 15% (v/v) FBS, 100 U/mL penicillin, 100 mg/mL streptomycin (Euroclone, Milan, Italy) and 2 mM L-glutamine (Euroclone, Milan, Italy) and maintained at 37 °C under humidified conditions and 5% CO₂. Cells were sub-cultured twice weekly, detached with Accutase (Euroclone, Milan, Italy) and centrifuged at 500× g for 10 min at room temperature. Cells were used for proteomics analysis at passage number lower than 10. Cell pellets were collected, washed in PBS, frozen in liquid nitrogen and stored at −80 °C.

We prepared and expanded MSCs as described previously (21 (link)). We flushed out bone marrow cells from tibias and femurs of 6-8-week-old C57BL/6J mice and kept the cells in plastic culture dishes in Mesencult medium (Stem Cell Technologies, Vancouver, BC, Canada) until a confluency of 80%. The cells were then detached using trypsin and 0.02% EDTA (Euroclone, Milan, Italy) and plated in 75 cm² flasks at the density of 4 × 10⁵ cells. After four to five passages in culture, mature MSCs were characterized by the expression of CD9, Sca-1, CD73, and CD44 and the lack of surface CD45, CD34, and CD11b. We verified their immunosuppressive activity in T-cell proliferation assays (21 (link)).

After 24 h, the exposure was stopped by collecting the exposure media and floating cells. Adherent cells were washed with ice-cold PBS, detached with trypsin/5 mM EDTA (EuroClone, Milan, Italy), and collected by centrifugation. The cell pellet was resuspended and washed twice in ice-cold PBS. Next, nuclear and cytosolic extracts were separated using different lysis buffers as described [53 (link)]. The protein concentration was determined using a bicinchoninic acid assay (QuantiPro^TM BCA Assay kit for 0.5–30 mg/mL protein, Merck, Darmstadt, Germany) following the manufacturer’s instructions. The assay was performed in triplicate for each experimental condition (n = 3).

Isopropyl β-d-1-thiogalactopyranoside (IPTG), sodium selenite (Na₂SeO₃), l-cysteine, ampicillin, chloramphenicol, riboflavin, lysozyme, Tris(hydroxymethyl) aminomethane (Tris), 1,4-Dithiothreitol (DTT), β-mercaptoethanol (βme), potassium phosphate, sodium chloride (NaCl), imidazole, flavin adenine dinucleotide (FAD), Auranofin were from Sigma-Aldrich. 2-methyl-2,4-pentandiol v/v 100% (MPD) from Molecular Dimension. EDTA was from Euroclone. Phenylmethanesulfonylfluoride (PMSF) was from ICN Biomedicals Inc. NADPH, Tetrasodium salt was from Roche and/or Sigma. 5,5′-dithiobis-(2-nitrobenzoic acid) (DTNB) and bovine pancreas insulin were obtained from Sigma. TRi-1 and TRi-2 were obtained from Oblique Therapeutics.

At the end of the exposure to modeled µg, medium was removed and cells were washed with Ca²⁺- and Mg²⁺-containing PBS (pH 7.4) (Euroclone S.p.A., Pero, Italy). Cells were then detached by Trypsinization (Trypsin 0.05%–EDTA 0.02%, Euroclone S.p.A., Pero, Italy) and counted with a Burker’s chamber under an inverted light microscope (Nikon, Amsterdam, The Netherlands). The same procedure was applied to 1× g control samples.

Negative cells, which contain tumour cells, were plated in 12-well plates at a concentration of 0.5–1 × 10⁶ cells/mL of CellGro SCGM (Cell Genix, Freiburg, Germany, cat# 20802-0500), supplemented with 20% foetal bovine serum (FBS) (Euroclone, cat# ECS0180D) and 0.1% gentamicin (Gibco, Life Technologies Limited, Paisley, UK, cat# 15750-037), and cultured in 25 cm² tissue flasks (Corning Stone, Staffordshire, UK) at 37 °C and 5% CO₂. Viable tumour cells attached to the flask within 12–24 h. At the first medium change, the cells were put into a fresh flask. The cells at 75% to 100% confluence were detached with 0.25% trypsin and 0.02% EDTA (Euroclone) in a calcium/magnesium-free balanced solution. The culture medium was changed twice a week and cellular homogeneity was evaluated microscopically every 24–48 h. Tumour cells derived from early passages (3–5) cultures were cryopreserved in 90% FBS and 10% DMSO (Alchimia, Ponte San Nicolò, Italy, cat# CRN 001-00) and stored in liquid nitrogen for further experiments. To confirm the neoplastic origin of the cultured cells, the cells underwent morphological and immunocytochemical analysis, as previously described [9 (link),26 (link)]. We were able to isolate and grow tumour cells from all 10 patients enrolled in this study.