D-Luciferin

Ⅰ. Matters needing attention

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.

一、注意事项

如果用于检测ATP，请戴上手套并使用不含ATP的高压灭菌水、试剂以及容器，以尽量减少所有可能的 ATP 污染源。

二、实验方法：

以下方案是钾和钾的示例钠盐制备。它适用于大多数细胞类型和体内动物使用。

本方案仅提供一个指导，应根据您的具体需要进行修改。

1、体外（细胞内）荧光成像

（1）将稳定表达荧光素酶的细胞接种在12孔板（2×10³/孔）中。

（2）使用无菌水制备100 mM荧光素原液，充分溶解后使用0.22µm滤膜过滤除菌（避光）。

（3）使用预热的培养基将母液稀释至浓度为0.5-1 mM的工作液

（4）从培养的细胞中吸出培养基，向细胞中加入荧光素工作液，并在成像前于37℃孵育细胞5-10分钟^[1]。

（5）使用一系列滤波器(520- 800nm)进行图像采集

2、体内荧光成像

（1）使用无菌D-PBS（不含Ca2+和Mg2+）配制D-荧光素钾盐原液（15mg/ml），充分溶解后使用0.22µm滤膜过滤除菌（避光）。

（2）成像前10-15分钟腹腔注射动物，给药剂量：75-150mg /Kg ^{[2] [3]}。

（3）使用生物发光成像对实验动物进行荧光成像

注意：应对每个动物模型进行荧光素动力学研究以确定峰值信号时间。

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.