Antibiotic antimycotic

Trans-mitochondrial cybrids and HEK293T cells were cultured at 37 °C and humidified 5% CO₂ in Dulbecco’s modified Eagle’s medium (DMEM; Lonza, Basel, Switzerland), supplemented with 10% (v/v) Foetal Bovine Serum (FBS; ThermoFisher Scientific, Carlsbad, CA, USA), 1 mM sodium pyruvate, 1× non-essential amino acids, 50 µg/mL uridine and 1× Antibiotic-Antimycotic (Euroclone, Milan, Italy).
For PCR-RFLP determination of the 3243A>G mutation load, total DNA was extracted from cybrids using the Wizard^® Genomic DNA Purification Kit (Promega, Madison, WI, USA), following manufacturer’s recommendations. The 3243 locus on the human mitochondrial genome was amplified using the following primers: Melas.FOR, 5′-CCTCGGAGCAGAACCCAACCT-3′; and Melas.REV, 5′-CGAAGGGTTGTAGTAGCCCGT-3′. After PCR reactions, the amplified products were ApaI-digested, fractionated on 1.5% agarose and stained with GelRed^® (Biotium, Fremont, CA, USA). The proportion of mutant (digested) to wild-type (undigested) mtDNA fragments was determined by ImageQuant TL 8.1 software (GE Healthcare Life Sciences, Pittsburgh, PA, USA).

The THP-1 monocyte cell line was bought from Resnova (Rome, Italy) and was cultured in RPMI 1640 medium [-] L Glutamine (Thermo Fisher Scientific, Waltham, MA, USA) with the addition of 2 mM L-Glutamine (Euroclone S.p.a, Pero, Italy) and 10% FBS (Euroclone S.p.a, Pero, Italy). Cell cultures were maintained at 37 °C and 5% CO₂, and the medium was changed twice a week. For all experimental purposes, differentiation of THP-1 monocytes into macrophages was carried out with complete RPMI 1640 medium containing 100 ng/mL of Phorbol 12-myristate 13-acetate (PMA) for a period of 24 h. Subsequently, a resting period of 72 h was allowed before treatments, in which adhered macrophages were cultured with complete RPMI 1640 medium [28 (link)]. Fibroblasts and NCTC L929 cell lines were also purchased from Resnova (Rome, Italy) and were cultured with DMEM medium (Euroclone S.p.a, Pero, Italy) with 10% FBS and 1% antibiotic–antimycotic (Euroclone S.p.a, Pero, Italy). Fibroblasts and macrophages were further treated with TNFα (10 ng/mL) and IFNg (50 ng/mL) for 6 h to induce inflammation.
Treatment with ADNVs fraction was performed by adding an amount of ADNVs solution mounting to a concentration of 8 × 10¹⁰ particles/mL to cultured cells. The control group was instead constituted by untreated cells.

Normal colon naive fibroblasts and tumour-associated fibroblasts (TAFs) were derived from the same patient diagnosed with colon adenocarcinoma. The naive fibroblasts were isolated from the normal colon segment approximately 20 cm from the tumour close to the edge of the isolated colon segment that was histologically free of neoplasia. The isolated tissues were washed rigorously in phosphate-buffered saline (PBS), an antibiotic-antimycotic (Lonza, Basel, Switzerland), gentamycin (Lonza) and cut into pieces for incubation overnight at 37 °C in αMEM (Euroclone, Milano, IT) with antibiotic-antimycotic and gentamycin in the presence of collagenase type II 150 U/ml (Gibco, Waltham, MA, USA). The next day, the undigested tissue pieces were removed by centrifugation, and the supernatant was plated on gelatine (1 g/l) pre-treated dishes in αMEM (Euroclone) supplemented with 10% defined FBS (GE-Healthcare, Chicago, IL, USA), non-essential amino acids (Euroclone), L-alanine-L-glutamine (Euroclone), penicillin/streptomycin (Euroclone), antibiotic-antimycotic, and gentamycin.