Ivis 100 system

For the in vitro BLI, cells were plated in triplicate in 24-well plates at a density of 1×10⁵ cells per well in normal growth medium, and kept under standard incubation conditions. After 24 hours, cells were washed with PBS and incubated with 250 µL of D-luciferin (0.3 µg/mL; Promega, Benelux, Leiden, The Netherlands). Cells were placed in the BLI chamber immediately for the acquisition of 1 minute scans.
Animals were anesthetized with 2% isoflurane in 100% oxygen, at a flow rate of 2 L/minute, after which D-luciferin, dissolved in PBS (15 mg/mL), was injected intravenously (126 mg/kg body weight). Images were acquired using an IVIS 100 system (Perkin Elmer). Consecutive 1 minute frames were acquired until the maximum signal intensity was reached. Each frame depicts the bioluminescence signal intensity as a pseudocolor image superimposed on the gray-scale photographic image. The data are reported as total photon flux (p/s) from a circular region of interest (ROI). BLI signal intensity was monitored over 8 days after xenograft generation.
For the quantification of BLI data in the mouse xenograft model, values from according ROIs measured with CLI were subtracted from the raw BLI ROI values to obtain specific BLI signal intensities.

For the hMultistem cells, eGFP expression was used for visual confirmation of transduction after which fLuc and HSV-tk expression were assessed. For fLuc expression, 100.000 cells were seeded in triplicate in a 24-well plate and were allowed to attach before BLI measurements were performed. An amount of 75 μg of D-luciferin (Promega, Madison, WI, USA) was added per well prior to BLI experiments using an IVIS 100 system (Perkin Elmer, Waltham, MA, USA). The temperature was maintained at 37°C. Scanning parameters included medium binning, f stop = 1, time = 10 s or 1 min. Data were analyzed by using the living image 2.50.1 software. Similarly, mCherry expression was used for the tumor cell lines in order to confirm transduction by fluorescence microscopy. BLI was performed using the rLuc substrate coelenterazine-h (Rediject Coelenterazine-h, Perkin Elmer, 0.25 μg/well).
HSV-tk expression was confirmed with a GCV killing experiment. Hereby, 20.000 cells were seeded in a 24-well plate and were allowed to grow. The following day, ganciclovir (Cymevene, Roche, Basel, Switzerland) was added in different concentrations (100 μM, 1 μM, and 0.01 μM) for four consecutive days, after which BLI was performed. Cells were subsequently collected and a BCA protein assay (Thermo Scientific, Rockford, USA) was performed.

The mice were imaged in an IVIS 100 system (PerkinElmer, Waltham, MA, USA). Anesthesia was performed in an induction chamber with 2% isoflurane (Halocarbon Products Corporation, River Edge, NJ, USA) in 100% oxygen at a flow rate of 1 L/min and maintained in the IVIS with a 1.5% mixture at 0.5 L/min. Since fur negatively influences BLI signals [31 (link)], the head of the mice was shaved before each imaging session. 126 mg/kg D-luciferin (Promega, dissolved in PBS (15 mg/mL)) was injected i.v. Immediately after injection, the mice were placed in the prone position in the IVIS and consecutive 1 min frames were acquired until the maximum signal, between 1 and 5 min after luciferin injection, was reached. The data are reported as the photon flux (p/s) from a 0.13 cm² square region of interest.