Living image 3

A431 cells (1 × 10⁶) were implanted s.c. into the dorsal space of 6-week-old female Hsd:athymic Nude-Foxn1nu mice. Tumor growth was monitored two times a week by measuring the diameter of the tumors with a calliper, and tumor volumes were calculated according to the formula: Volume = (D × d2/2), where D is the longest axis of the tumor and d is the shortest of a prolate ellipse. When tumor volume reached about 0.180 cm³, mice were randomly allocated to different treatment groups (n = 3/group) and i.v. injected with PBS or NIR-labeled antibody solution (1 mg/ml) in PBS. Mice were imaged under anesthesia at 24 h under the IVIS Spectrum CT in vivo imaging system (Xenogen) at the indicated wavelengths and were analyzed using the Living Image 3.2 software (PerkinElmer). The images were analyzed by identifying 3 ROIs within the tumor and from surrounding regions (normal tissue). The T/N ratio was calculated by dividing the mean values of the identified ROIs. Fluorescence intensity of all the images are reported as photons per second per centimeter squared per steradian (p/s/cm²/sr).

Tokeoni (a luciferin analog) was dissolved in saline solution (60 mmol/L) and injected intravenously into the monkeys (75 nmoL/g, Tokeoni). Immediately after the addition of Tokeoni, the monkeys were imaged using an IVIS Spectrum (PerkinElmer) continuously. Bioluminescence was quantified using region of interest (ROI) analysis of the individual wells. The average signal, expressed as the total number of photons emitted per second (photons/s), from each of the wells was calculated using the Living Image 3.2 software (PerkinElmer).
All imaging experiments required anesthesia in monkeys. Prior to anesthesia, a 12-h fasting period was implemented, and intramuscular injections of atropine sulfate solution (0.05 mg/kg) and ZoletilTM (0.05 mg/kg) were administered. The experiments were initiated once the monkeys were observed to be in a normal physical condition after the injections. Following the completion of the experiments, intramuscular injections of xylazine hydrochloride solution (0.025 mL/kg) were administered. This approach typically maintained the anesthesia state for approximately one hour. Imager settings (Table S2).

Ninety-six-well black color plates with transparent bottom (Costar) and a plate reader (SpectraMax, Molecular Devices) were used for in vitro fluorescence imaging. For in vivo experiments, mice carrying 4T1 tumors were injected with CNOB (3.3 mg/kg iv) prior to imaging. The images were acquired by IVIS Spectrum (Perkin Elmer Inc.) and quantified by Living Image 3.2 software (Perkin Elmer Inc.). The exposure time for photography was 1 s. A standard curve was constructed in vitro based on the quantified photon intensity at various MCHB concentrations (0, 1.5, 15, 150, 1500 μM). Bioluminescence was measured using the same instrument. We note that the IVIS instrument is widely used for quantitative fluorescence/bioluminance imaging, as it possesses a built-in calibration system. As already stated, this permitted generation of a standard curve that linearly related MCHB photon yield with its concentration. The imaging experiments were performed at least four times.

For visualization of photon emission from the bioluminescent strain S. aureus Newman lux, an in vivo imaging system (IVIS Lumina II; Perkin Elmer, Waltham, MA, USA) was used. Mice were anesthetized with 2% isoflurane and imaged with following parameters: exposure, 120 s; F stop, 1; excitation, block; emission, open; field of view (FOV), D; and height, 1.5 cm. The bioluminescence signal was measured for a defined region of interest with standardized size for all mice and time points at the site of infection. The average radiance values were analysed using the corresponding Living Image 3.2 software (Perkin Elmer).

Mice received 1 × 10⁶ MSCs expressing firefly luciferase and green fluorescent protein^{24 (link)} via tail vein injection. Ten minutes prior to imaging, mice were i.p. injected with 200 μL D-luciferin (15 mg/ml in PBS, VivoGlo Luciferin; Promega, USA. Mice were imaged under anaesthesia with 2.5% isoflurane in oxygen, or euthanised for open-chest and direct lung imaging. Imaging was performed on the Xenogen IVIS 200 or Spectrum (Perkin Elmer, USA) and analysed using the Living Image 3.2 Software (Perkin Elmer, USA). For AM depletion, mice received 60 μl of clodronate-encapsulated liposomes (5 mg/ml clodronate in liposomal structures containing 18.8 mg/ml phosphatidylcholine and 4.2 mg/ml cholesterol in PBS; Encapsula NanoSciences) via intranasal administration under light anaesthesia on days −4 and −2 prior to MSC injection on day 0.

An IVIS Spectrum Imaging System (Perkin Elmer Inc) was used for in vivo and ex vivo fluorescence imaging. For in vivo imaging, mice were anesthetized with a 4% rate of isoflurane (IsoVet Braun) during the induction, and 2% during the maintenance period (scanning time). For ex vivo imaging, mice were sacrificed by CO₂ exposure in a euthanasia chamber, and organs and tumor xenografts were analyzed immediately after harvesting. Rhodamine B was detected using excitation wavelength of 535 nm and emission wavelength of 580 nm. Indocyanine green was detected using excitation passband of 710–760 nm and emission passband of 810–875 nm. Doxorubicin was detected using excitation wavelength of 430 nm and emission wavelength of 600 nm. Fluorescence imaging quantification was performed by Living Image 3.2 software (Perkin Elmer Inc). A region of interest area (ROI) was drawn over the fluorescent signal either in lung or xenograft tumor samples in order to quantify the release of the fluorophores. Fluorescence activity is measured in photons per second per square centimeter per steradian (p/s/cm²/sr).

To study the effect of c-di-AMP on the induction of IFN-β genes C57BL/6 IFN-ββ-luc reporter mice (see also paragraph “Mice”, above) were used. The conditional reporter mice received c-di-AMP by the i. n. route at a concentration of 5 µg per mouse. IFN-β gene induction was analyzed by measuring luciferase activity as a reporter by in vivo imaging. To this end, mice were injected intravenously with 150 mg/kg of D-luciferin firefly (Synchem, Felsberg/Altenburg, Germany) dissolved in PBS at time points 0, 3, 6, 12 and 24 h after administration of c-di-AMP. Mice were anesthetized with Isofluran (Curamed, Karlsruhe, Germany) and monitored using the IVIS 200 imaging system (CaliperLS, Perkin Elmer Inc., Waltham, Massachusetts, USA). Photon flux was quantified with the LivingImage 3.2 Software (CaliperLS, Perkin Elmer Inc., Waltham, Massachusetts, USA) and is expressed in photons/s/cm²/steradian.