Living image software package

Photon emission of luminescent P. aeruginosa (PAK’ΔloxA-luxCDABE) in the mouse was measured using the IVIS Lumina XR system (Perkin Elmer), which includes an IVIS charge-coupled device camera coupled to the LivingImage software package (Perkin Elmer). Analysis of photons was done under isoflurane inhalation anesthesia. A digital false-color photon emission image of the mouse was generated, and photons were counted using a 3-min acquisition time using the following settings: Medium binning (M), Field Of View: 12.5 cm (D), f1. Image analysis and luminescence quantification have been performed with LivingImage software (Perkin Elmer). Region Of Interest (ROI) were defined as area corresponding to the surface of the chest encompassing the whole lung region after 3 min acquisition time.

All animal protocols were approved by the Institutional Animal Care and Use Committee of Shandong Cancer Hospital and Institute, China. A subline of HNE2 cells called HNE2/luc was used, which was transfected with the firefly luciferase gene. BALB/c-nu mice (4–6 weeks of age, female, from Beijing HFK Bioscience Co., Ltd., China) were subcutaneously injected into the flank with 3 × 10⁶ HNE2/luc cells in 100 μL PBS. The mice were housed in laminar flow cabinets under specific pathogen-free conditions. Seven days after the implantation, efficient tumor volume was confirmed in all animals via noninvasive whole-body bioluminescent imaging. For this purpose, mice were intraperitoneally injected with 50 mg/kg D-luciferin (Perkin Elmer, MA, USA) and imaged using the Xenogen IVIS Spectrum Imaging System (Caliper/Perkin Elmer). Then mice were randomly divided into five groups, five in each group, and treated once a day for 5 days with the following: Control (DMSO), TMZ, POH, TMZ plus POH, and TMZ-POH. Mice were imaged twice per week. Images were analyzed by region-of-interest (ROI) analysis using the Living Image software package (Caliper/Perkin Elmer, MA, USA) to quantitate the tumor volume. Mice were sacrificed and examined for the growth of tumors 17 days after the cessation of drug treatment.

Xenografts were established as described above. Two weeks after the injection of cells, animals were distributed into treatment groups based on tumor size which was determined by bioluminescence imaging (IVIS Spectrum system equipped with Living Image software package, Perkin Elmer). Animals either received intraperitoneal injections of a mixture of 0.1% DMSO in saline as vehicle or 8 mg/kg ACF in saline (Sigma #01673) for a total of 15 injections. Tumor size was measured using bioluminescence imaging. After sacrifice, xenografts were removed and sectioned into two parts. One part was snap frozen on dry ice for molecular analyses. The second part was dissociated into single cell suspensions. Roughly one-half was immediately analyzed by flow cytometry to detect the presence of ACF in cells (as described above). The remaining cells were cultured for three days and then challenged with vehicle or ACF in vitro. Total viable cell mass was determined by an Alamar Blue growth assay (see below).

To determine the biodistribution of H1/pORF-Luc nanoparticles after their systemic administration, in vivo optical imaging of living Balb/c mice was performed using an IVIS 100 series imaging system (PerkinElmer, Waltham, MA, USA). Briefly, at the time point of detection, the mice were anesthetized, and D-luciferin (Xenogen) at a dose of 150 mg per gram of mouse body weight were injected intraperitoneally 5 min before the images were taken. The images were obtained at exposure times of 1 to 10 min, depending on the intensity of the emitted photons. The total photon flux (photons/second) in a region of interest (ROI) was quantitated using the Living Image^® Software package (version 4.3.1 PerkinElmer, Waltham, MA, USA).

Optical images were acquired on an IVIS Lumina XR imager (PerkinElmer, USA) with an exposure time of 1 min, an open aperture and an open emission filter. A photographic image was acquired for anatomical co-registration of the signal. Luciferase expression of each set of cells was confirmed using BLI. Isolated MSCs (0.6 × 10⁵ − 1 × 10⁶) were placed into a six-well plate and imaged for 1 min after the addition of 10 μl of d-luciferin (30 mg/ml). The MSC recipients were examined before and after intravenous administration of d-luciferin dissolved in sterile PBS (at a dose of 15 mg) in a time series lasting 14 min. The bioluminescent colour-coded images were superimposed on the photographic images and analysed using the Living Image software package (PerkinElmer, USA). Signal intensity was assessed as photons per second per square centimetre per steradian (p/s/cm²/sr) from the area containing the scaffold. The area under the curve (AUC) was calculated from each time course in order to minimise the variability in d-luciferin administration among the measurements. The bioluminescence examination was performed at the same time points as the MRI experiments; one animal was monitored for 16 months.