CycLuc1

CycLuc1 based-bioluminescence imaging experiments in living mice:

1.Prepare the CycLuc1 solution:

The 50μmol/L-5mmol/L CycLuc1 working solution is prepared in PBS buffer (pH7.4). After the configuration is completed, filtration sterilization is carried out using a 0.22μm filter membrane.

2. Feeding of mice:

Pathogen-free BALB/c, FVB/N, C57BL/6 and luciferase-expressing transgenic mice (FVB-Tg(CAG-luc,-GFP)L2G85Chco/FathJ) were provided access to food and water ad libitum.

3. Tumor cell inoculation:

Subcutaneous inoculation of mice involved 1×10⁶ luciferase-expressing CMT-64 or DB7 cells suspended in serum-free RPMI medium. The tumors achieved their growth phase over a span of 1–2 weeks before imaging procedures commenced. To establish 4T1 tumors, aliquots of the cells were mixed with the mixture of cells with Matrigel at 1:1 volume ratio (5×10⁵ cells with 50μl serum-free DMEM medium combined with 50μl Matrigel), were implanted into second or fourth mammary fat pads on the left or right sides. Mice received intraperitoneal injections 8 days following engraftment. After administering 100μl of either D-luciferin solution or CycLuc1 at concentrations ranging from 50μmol/L to 5mmol/L the mice received imaging 10 minutes following the injection.

4. Bioluminescence imaging:

Administer isoflurane anesthesia (2% concentration in 1L/min oxygen) to the animals before capturing bioluminescence images using the IVIS Lumina system. The imaging protocol involved capturing images every 2 minutes throughout a 30-minute period, each with 10 seconds of exposure time. Acquire images of the mice at both 1 hour and 2 hours post-injection with each image exposed for 10 seconds. Images were analyzed using Living Image software. Outline regions of interest (ROIs) around each cellular cluster to measure the total photon count within each defined area. All images maintained the same ROI size.

Reference:
[1] Evans M S, Chaurette J P, Adams Jr S T, et al. A synthetic luciferin improves bioluminescence imaging in live mice[J]. Nature methods, 2014, 11(4): 393-395.