Living image 4

Mice were injected i.p. with 150 mg/kg XenoLight d-luciferin – K⁺ Salt (PerkinElmer, U.K.). Mice were left for 10 min prior to the induction of anesthesia using isoflurane and bioluminescence measurement using an IVIS Lumina II Imaging System (PerkinElmer). Average radiance for areas of interest in bioluminescence images were calculated using Living Image 4.2 Software (PerkinElmer). For an in vitro measurement of bioluminescence using cultured cells, cells were transferred to black 96-well tissue culture plates before the addition of d-luciferin at a final concentration of 150 μg/ml. Cells were incubated for 10 min before imaging.

Stable cell lines expressing the TOP-FLASH reporter were generated by transducing
cells with 7TFP recombinant lentiviruses (Fuerer
and Nusse, 2010), and luciferase assay was performed as previously
described (Singh et al., 2012 (link)). Briefly,
Rediject D-Luciferin Ultra (Perkin Elmer) was added in 0.2 ml fresh media
(1–200 dilution) to each well of cells in a 96-well plate and incubated for 15
min at 37°C. Luciferase activity was imaged with the IVIS Lumina II In Vivo
Imaging System (Perkin Elmer). The radiance of each well was determined using Living
Image 4.2 software (Perkin Elmer), background corrected by subtracting the mean
signal from empty wells and normalized both to the relative cell number of each well
as determined by Syto60 assay and the resulting normalized mean value of untreated
wells.

Tumor growth and spread after tail vein injection was monitored intra-vitally once per week for a total of 5 weeks. Luciferin (Promega, Madison, WI) was prepared at a final concentration of 300 mg/ml. After induction of anesthesia as described above, 300 μL of Luciferin was injected intraperitoneally per mouse and allowed to distribute through the body of the animal for 15 min before imaging. Imaging was performed using an IVIS Spectrum pre-clinical in vivo imaging system (PerkinElmer, Waltham, MA) under continued anesthesia. Images were taken after 10 s, 1 min and 3 min each for supine and prone position. Bioluminescence was quantified using the Living Image 4.2 software (PerkinElmer).

MDA-MB-231 expressing Luciferase were plated in 24 well plates and left to adhere for 24 h. The next day, the indicated concentration of drugs were added to the wells and Beetle Luciferin (Promega) was used to quantify the cells using the Xenogen IVIS 200 with Living Image 4.2 software (PerkinElmer). For proliferation, the plates were imaged after 24 h, 48 h and 72 h of treatment and for the viability assay, the plates were imaged after 24 h.

Animal experiments were performed in compliance with the guidelines of the Institute for Laboratory Animal Research, National Cancer Center Research Institute (Number: T12-004). Six- to seven-week-old male C.B-17/Icr-scid/scidJcl mice (CLEA, Tokyo, Japan) were used in the experiments. Intrathoracic injections were given following a previous protocol with minor modifications [20 (link)]. The cells were resuspended in PBS as a half volume of inoculum and diluted with an equal volume of growth factor-reduced Matrigel (356231; BD Biosciences). For in vivo imaging, the mice were administered 150 μg/kg D-luciferin (Promega, Madison, WI) by intraperitoneal injection. Ten minutes later, photons from the whole body of the animal were counted by measuring the bioluminescence with an IVIS imaging system (PerkinElmer) according to the manufacturer's instructions. The data were analyzed using the LIVINGIMAGE 4.2 software (PerkinElmer). Lung cancer development was monitored twice a week in vivo by bioluminescent imaging. In the repeated administration study, the treatment (2.5 μg/kg of CDDP) was performed on days 7, 14, and 21 (once a week for three weeks, three treatments total).