D-Luciferin (sodium salt)

Ⅰ. Matters needing attention

1. D-luciferin is easily soluble in aqueous buffer solution (pH 6.1-6.5), and the solubility can reach up to 100 mM. For example: Use sterile D-PBS (without Ca2+ and Mg2+) to prepare D-luciferin potassium salt solution (15mg/ml), and filter it with a 0.22µm filter membrane (protect from light). D-luciferin solution is recommended to be prepared and used immediately. It can also be frozen and stored at -20°C or -80°C after aliquoting to avoid repeated freezing and thawing. Melt at 4°C before use, and equilibrate to room temperature before experiment (protect from light)

2. If used to detect ATP, please wear gloves and use ATP-free autoclaved water, reagents and containers to minimize all possible sources of ATP contamination.

Ⅱ. Experimental method:

The following scheme is an example sodium salt preparation of potassium and potassium. It is suitable for most cell types and in vivo animal use.

This protocol provides a guide only and should be modified according to your specific needs.

1. In vitro (intracellular) fluorescence imaging

(1) Seed cells stably expressing luciferase in a 12-well plate (2×10³/well).

(2) Use sterile water to prepare 100 mM fluorescein stock solution, and after fully dissolved, use a 0.22 µm filter membrane to filter and sterilize (protect from light).

(3) Use pre-warmed medium to dilute the stock solution to a working solution with a concentration of 0.5-1 mM

(4) Aspirate the medium from the cultured cells, add fluorescein working solution to the cells, and incubate the cells at 37°C for 5-10 minutes before imaging^[1].

(5) Use a series of filters (520- 800nm) for image acquisition

2. In vivo fluorescence imaging

(1) Use sterile D-PBS (without Ca2+ and Mg2+) to prepare D-luciferin potassium salt stock solution (15mg/ml), and after fully dissolved, use a 0.22µm filter membrane to filter and sterilize (protect from light).

(2) Inject animals intraperitoneally 10-15 minutes before imaging, at a dosage of 75-150mg/Kg ^{[2] [3]}.

(3) Fluorescent imaging of experimental animals using bioluminescent imaging

Note: Fluorescein kinetic studies should be performed for each animal model to determine peak signal time.

References:

[1]. Giuseppe Meroni, et al. D-Luciferin, derivatives and analogues: synthesis and in vitro/in vivo luciferase-catalyzed bioluminescent activity. ARKIVOC 2009 (i) 265-288.

[2]. Wentian Zhang,et. Dual inhibition of HDAC and tyrosine kinase signaling pathways with CUDC-907 attenuates TGFβ1 induced lung and tumor fibrosis. 2020 Sep 17;11(9):765. doi: 10.1038/s41419-020-02916-w.

[3]. Senlin Li,et. Concurrent silencing of TBCE and drug delivery to overcome platinum-based resistance in liver cancer. 2023 Mar;13(3):967-981. doi: 10.1016/j.apsb.2022.03.003. Epub 2022 Mar 12.