Minimum Essential Medium Eagle

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	enriched with 10% Fetal Bovine Serum (FBS) (Sigma Aldrich) and 2 mM l-glutamine (Sigma Aldrich)

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enriched with 10% Fetal Bovine Serum (FBS) (Sigma Aldrich) and 2 mM l-glutamine (Sigma Aldrich)

Publication protocol

Vero cells (African green monkey kidney, IZSLER Cell Bank code BS CL 86) were received from ATCC (American Type Culture Collection) at p124 and grown in MEM culture medium (Eagle’s Minimum Essential Medium in Earle’s BSS) (Sigma Aldrich, Milano, Italy) enriched with 10% Fetal Bovine Serum (FBS) (Sigma Aldrich) and 2 mM l-glutamine (Sigma Aldrich). From p127 they were grown concurrently in serum-free medium. XerumFree™ XF205 Medium Supplement (TNC BIO, Eindhoven, Holland) was gradually added to the culture medium in substitution of FBS, starting from 50% XerumFree-supplemented medium/50% FBS-supplemented medium, according to manufacturer instructions. Cultures adapted to serum-free conditions were amplified in medium supplemented with Epidermal Growth Factor (EGF; 12.5 µg/L, Sigma Aldrich) and insulin (1.25 mg/L, Sigma Aldrich). At each passage, cells were mechanically scraped and incubated in a mixture of 75% fresh medium and 25% conditioned medium, collected during the previous passage.

HEp-2 (Human larynx epidermal carcinoma, BSTCL 23) and MRC-5 cells (Human foetal lung, BSCL 68) (both from the IZSLER biobank), were grown in MEM culture medium supplemented with 10% FBS and 2 mM l-glutamine. These cell lines were used respectively as positive and negative control in tumorigenicity and transformation assays.

A primary cell culture (passage 3) of a normal adult African Green Monkey Kidney (AGMK, Cercopithecus aethiops) was kindly provided by Dr. Brandini (Novartis Vaccines Italia, Siena, Italy) and grown in MEM culture medium enriched with 10% FBS, 1% Penicillin/Streptomycin and 2 mM l-glutamine. This cell line was used as reference in the in vitro transformation assay and karyotype analyses.

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