Vivoglo

D-luciferin (150 mg/kg VivoGlo, Promega, # P1043) was injected i.p., mice anesthetized with Isoflurane, and images taken 5 minutes after injection using a Xenogen IVIS-100 (Caliper).

Experimental lung metastasis experiments were performed as described previously [17 (link), 42 (link)]. C.B-17/lcrHsd-Prkdcscid mice were purchased from Japan SLC (Hamamatsu, Japan). All experiments were approved and performed according to the guidelines of the Care and Use of Laboratory Animals of the University of Toyama. Cells were inoculated intravenously (2 × 10⁶ cells/200 µL PBS/mouse) into mice and 200 µL of luciferin (1.5 mg/mL [VivoGlo; Promega, Madison, WI, USA]) was intraperitoneally injected into mice at 24 h after the tumor inoculation. After 20 min, the lungs were removed to subject bioluminescent assay by using an in vivo imaging system (IVIS Lumina II, Caliper Life Sciences, Hopkinton, MA, USA). The data are presented as the mean luminescence ± SEM.

In vivo follow-up was performed after tumor inoculation and carried over the 4 weeks following surgery or phantom operation. Mice were anesthetized with 4% of isoflurane vaporized in 2 L/min O₂ and then maintained with 2% isoflurane in 0.3 L/min O₂ per mouse. Before imaging, mice were shaved to decrease the light absorption and scattering of animal fur. Each animal received s.c. 150 mg/kg body weight of a 20-mg/ml solution of D-luciferin in a 20-mg/ml solution in NaCl 0.9% (VivoGlo, Promega). Mice were imaged in a Photon Imager Optima (Biospace Lab, France) that dynamically counted the emitted photons for at least 25 min. Image analysis was performed with M3Vision software (Biospace Lab). ROIs were drawn on the mice abdomen in the liver area and signal intensities were quantified individually for a time lapse of 5 min corresponding to the maximum signal intensity plateau.

Cell numbers were measured by bioluminescence imaging (BLI), CellTiter Glo (Promega, Madison, WI), or RealTime Glo (Promega) assays, according to the manufacturer’s instructions, and read on a GLOMAX microplate reader (Promega). Cell cycle analysis was measured with DAPI (0.5 µg/ml) and Ki67 staining (Alexa Fluor 647 Ki67 antibody, 350510, BioLegend). Apoptosis was measured using an annexin V/APC and DAPI Kit (BioLegend); total apoptotic cells were defined as annexin V⁺/DAPI⁺+annexin V⁺/DAPI^- populations. Data were acquired on a Miltenyi MACSquant flow cytometer and data analysis was performed using FlowJo software (BD Life Sciences). For BLI in vitro imaging of luciferase expressing cells, sterile luciferin (10 µL/well from a 7.5 mg/mL stock, VivoGlo, Promega) is added to white, 96 well plates of cells, given 5 min to reach equilibrium, and read in a GLOMAX microplate reader (Promega). For flow cytometry, a minimum of 10,000 events was collected and gated off forward and side scatter plots.

pRBC sequestration was measured by whole body imaging of mice injected with P. berghei-ANKA line expressing luciferase essentially as described by Claser et al.46 (link). The bioluminescence images were visualized by an intensified-charge-coupled device (I-CCD) photon-counting video camera from the in vivo Imaging System IVIS Lumina 200 (Xenogen)8 (link). C57BL/6 mice were infected with 3 × 10⁶P. berghei ANKA transgenic parasite line expressing GFP-Luciferase. On the specified days post infection, animals were injected intraperitoneally with D-Luciferin substrate (promega, VivoGlo^™) and checked for bioluminescence signal after 10 min in the whole body and head of animals. Whole body and head imaging were carried out with 21.8 and 4 cm FOV, respectively and medium binning factor. The exposure time was varied between 50 and 300 sec depending on the intensity of the signal. In another set of experiments, animals were sacrificed, whole body perfusion performed, brains and other organs were removed and used for ex vivo imaging. Average radiance (p/sec/cm²/sr) values were calculated for region of interest (ROI) using Living Image®4.3.1(64 bit) software for experimental and control animals. Background values obtained from uninfected mice injected with luciferin were subtracted and plotted using GraphPad Prism 5 software.