D luciferin potassium

All animal experiments were performed following instructions and permissions of the ethical committee of Beijing Tiantan Hospital. Female Balb/c nude mice (5-week-old) were purchased from Charles River Laboratories, Beijing, China. The subcutaneous tumor models were established by injecting 5×10⁶ cancer cells dissolved in 100μL of PBS subcutaneously into the right shoulder. After 3 to 4 weeks, while tumors grew into 6-8mm, animal models were used for in vivo imaging. Brain metastatic tumor models were constructed by stereotactic injection of A549 (5×10⁵ cells dissolved in 5 μL) into the right hemisphere using the following coordinates: 1mm posterior to the bregma, 2mm lateral to the midline, and 3mm depth into the brain surface. Tumor growth was monitored by bioluminescence imaging (IVIS Spectrum, PerkinElmer), after intraperitoneal injection of D-luciferin Potassium (PerkinElmer, 15mg/mL, 0.15mg/g), and the total photon flux (photons/sec) was quantified by Living Image (v4.5.5, PerkinElmer).

Bioluminescence imaging was performed as described by Contag et al.^{23 (link)}. Briefly, D-Luciferin Potassium (Perkin Elmer, Waltham, MA) was dissolved in PBS at 30 mg/ml. Mice were injected subcutaneously with a dose of 150 mg/kg while anesthetized using 2–3% isoflurane in 2 L/min of oxygen. Mice were imaged 15 minutes post D-Luciferin injection under anesthesia using an IVIS Spectrum system (Perkin Elmer, Waltham, MA). The field of view was set to 25.6 cm, at medium sensitivity, and integration times ranged from 1 second to 2 minutes depending on signal intensity. Images were analyzed using Living Image 4.4 software (Perkin Elmer, Waltham, MA). Regions of interest were manually selected, and the output signal intensity, total flux, was expressed as photons per second.

For prophylactic vaccination, the mice (n = 15) were randomly assigned to 3 groups: Group 1 (n = 5) served as the control group and was injected with PBS. Group 2 (n = 5) was injected with 2 × 10⁶ of 4T1-luc2 cells that had been put through freeze and thaw (F/T) cycles, where the cells were first frozen in liquid nitrogen for 90 s, then thawed at 4 °C for 4 min, and this process was repeated 4 times. Group 3 (n = 5) was injected with 2 × 10⁶ of 4T1-luc2 cells that had been treated with crassolide (25 µM) for 24 h. Seven days post-vaccination, all four groups of mice were orthotopically implanted with 5 × 10⁵ live 4T1-luc2 tumor cells in 100 µL of PBS into the mammary fat pad. The tumor size was observed and measured three times a week and bioluminescence imaging of tumors was monitored once a week. Tumor size was measured by caliper measurements, and tumor volume calculation was used following formula: 0.5 × length (mm) × width² (mm²). Bioluminescence imaging of tumors was monitored using a noninvasive in vivo imaging system. The mice were injected intraperitoneally with 15 mg/kg of D-luciferin potassium (PerkinElmer, 122799) in PBS and were left for 5 min before imaging. The test mice were then anesthetized with 2.5% isoflurane using the XGI-8 Gas Anesthesia System (PerkinElmer, Boston, MA, USA), and the XENOGEN IVIS 50 (PerkinElmer, Boston, MA, USA) was applied.