*4.13. Animal Hosting, Inoculation and Monitoring*

Homozygous female BALB/c nude J:NU (JAX stock number 007850) mice were purchased from Jackson Laboratories (Bar Harbor, ME, USA). Mice were 4 weeks old at arrival, and they were hosted in an IVC system (Smart Flow AHU, Tecniplast) with autoclaved rodent bedding hosting 3–4 mice per cage. Mice were fed with autoclaved rodent food and water ad libitum. The inoculation of A549 tumor cell line was performed by the subcutaneous injection of 5 <sup>×</sup> <sup>10</sup><sup>6</sup> cells/mouse in 100 uL of fetal bovine serumfree medium (RPMI added of 10% FBS, 1% penicillin-streptomycin, 1% glutamine) into the right shoulder. The cell line was found to be free of mycoplasma contamination, as visually judged by microscopic inspection and by regular 40 ,6-diamidino-2-phenylindole (DAPI) staining of the cell cultures. Inoculation was carried out at 5 weeks of age after one week in the facility for acclimatization purposes. Mice were monitored every 2–3 days by measuring weight and tumor dimensions with an electronic caliper. For this purpose, two perpendicular dimensions were noted and volumes of the tumors were calculated with the formula 0.5 <sup>×</sup> <sup>L</sup> <sup>×</sup> <sup>W</sup><sup>2</sup> , where L is the measurement of the longest axis and W is the measurement of the axis perpendicular to L, in millimeters [16].
