Xenogen ivis 100

Metastatic disease burden and spread were quantified using weekly BLI on a Xenogen IVIS 100 in vivo imaging system (Perkin Elmer). The mice were subcutaneously injected with 150 mg/kg D-luciferin (50 mg/mL) and imaged 15 min post injection in the supine position. Living Image software was used to quantify whole body disease burden. The data were then plotted and analyzed using GraphPad Prism 8. GraphPad Prism 8 was also used to generate survival plots and statistical analyses. The Log-rank (Mantel-Cox) test was used for survival analysis.

Biodistribution studies of DiR’-labeled lipoplexes were carried out according to approved protocols (protocol 32/2011 CEASA University of Padua and 318/2015 Italian Ministry of Health to L.S). 10 male non-consanguineous CD-1 mice of 12 weeks old were maintained in a temperature (22ºC) and humidity (30–50%) controlled animal care facility with a 12 h light/dark cycle and free access to water and food. After intraperitoneal (N = 3), intracardiac (N = 3) or intramuscular administration (N = 3) of DiR’-labeled lipoplexes (400 µg/per animal), mice were anesthetized with a mixed solution composed of 15 µL of rompun (Xylazine, 20 mg/mL) and 25 µL of virbac zoletil (Tiletamine/zolazepam, 10/10 mg/mL). The biodistribution of DiR- labeled lipoplexes at different time intervals, was then determined on a Xenogen IVIS-100 (Perkin-Elmer) using the Cy5.5 filter (675/694 nm). After treatment, animals were sacrificed in a CO₂ chamber with gradual CO₂ and O₂ flux and by cervical dislocation. Subsequently, tissues from brain, spleen, liver, lungs, kidney, muscle and heart were dissected, washed with saline solution and frozen in liquid nitrogen for Western Blot analysis and RT-PCR.

T cell expansion was analyzed on the basis of luciferase signal intensity. Briefly, mice were i.p. injected with luciferin (200 ng/g body weight) and euthanized 7 minutes later. The organs were then imaged for 30 seconds using a Xenogen IVIS 100 (PerkinElmer). The imaging data were analyzed with Living Image Software (PerkinElmer) and are presented as photons per second.

Xenogen IVIS-100 (PerkinElmer) was used for in vivo imaging. Mice were injected intraperitoneally with VivoGlo luciferin, in vivo grade: P1042(Promega) (150 mg/kg). After 10 min, mice were anesthetized with 2% isoflurane, and then they were transferred into Xenogen IVIS-100. All mice were imaged twice weekly. Living Image (PerkinElmer) was used for analysis. All imaging processes followed manufacturer’s instructions. To measure BLI signal in the left, center, and right chest, a rectangular box capturing the chest cavity of each mouse was divided into thirds of equal area that correspond to left, center, and right. Comparisons between sets of experimental mice were made using BLI quantitation made on the same day, during the same imaging session.