Samples

We are currently working with Addgene, a nonprofit plasmid repository, to deposit frequently requested plasmids. Please see our page on Addgene:

addgene

If you don’t see your wanted plasmids on Addgene or if you need other types of materials, please contact Dr. Ai (huiwang.ai at virginia.edu).

Practical Notes for teLuc-DTZ and Antares2-DTZ (PDF)

There is considerable interest in adapting teLuc-DTZ or Antares2-DTZ for various biological and biomedical studies. The plasmids are now available from Addgene. If you need a small-amount DTZ sample, you may contact Dr. Ai. You may also purchase DTZ from Haoyuan Chemexpress Co., Ltd. (We do not make any profit by suggesting this company, but we have tested their DTZ compound). DTZ should be shipped in nitrogen-flushed dark-glass bottle, or on dry ice if oxygen is not completely removed. For long-term storage, DTZ should be kept as solid in -80°C freezers.

To make a stock solution for DTZ, first, a premixture is prepared by dissolving 17.6 mg of L-ascorbic acid and 143 mg 6-aza-2-thiothymine in 10 mL ethanol and 10 mL 1,2-propanediol; next, 1 mg of DTZ is dissolved in 88 uL of the premix, resulting in a 30 mM DTZ stock solution containing 5 mM L-ascorbic acid (you may increase the DTZ amount to make up to 100 mM stock). The stock solution should be stable for a few months in -80°C freezers. It may also be aliquoted to 10-20 uL each at -80°C for the convenience of use. We want to note that this new formulation greatly enhances substrate stability, compared to the conventional acidic alcohol solution.

To use DTZ for animal studies, 10 uL of the stock solution is further diluted with 90 uL of another prexmiture containing 9% glycerol, 11% hydroxypropyl-β-cyclodextrin, and 39% PEG 400 in water right before injection. This ready-for-use solution can sit on ice for a couple of hours. The suggested dosage is 0.3 to 1 umol DTZ for each ~ 20 g mouse.

Although cautions have to be made to maintain the in vitro stability of DTZ, background bioluminescence is generally not an issue for in vivo applications of DTZ. Background bioluminescence from DTZ is negligible compared to real signals. We have examined this with untreated blank BALB/c mice, BALB/c mice transfected with empty vectors, and BALB/c mice injected with cells containing no luciferase. When exogenous cells expressing teLuc were injected into immunocompetent BALB/c mice, we observed fast clearance. We expect that teLuc in dead cells or released from dead cells is still enzymatically active, since teLuc is independent of ATP and highly resistant to denaturation and unfolding. When comparing results with firefly luciferase or its derivatives, please note that firefly luciferase may only function in live cells where there are enough ATP.

Although DTZ has not yet been frequently used for animal imaging because our paper was only published several months ago, there are a plethora of literatures which used DTZ analogs, including CTZ and furimazine, for in vivo imaging. You may refer to these publications to better understand the potential of teLuc-DTZ and Antares2-DTZ for in vivo imaging. We select a few papers below:

Bhaumik S, Gambhir SS. Optical imaging of Renilla luciferase reporter gene expression in living mice. Proc Natl Acad Sci U S A. 2002, 99(1):377-82.
Loening AM, Wu AM, Gambhir SS. Red-shifted Renilla reniformis luciferase variants for imaging in living subjects. Nat Methods. 2007, 4(8):641-3.
Saito, K, Nagai T, et al., Luminescent proteins for high-speed single-cell and whole-body imaging. Nat Commun. 2012, 3: 1262.
Chu J, Lin MZ, et al., A bright cyan-excitable orange fluorescent protein facilitates dual-emission microscopy and enhances bioluminescence imaging in vivo. Nat Biotechnol. 2016, 34(7):760-7.
Franz X Schaub, Antonio L Amelio, et al., Fluorophore-NanoLuc BRET reporters enable sensitive in vivo optical imaging and flow cytometry for monitoring tumorigenesis. Cancer Res. 2015, 75(23):5023-33.