Natural substrate of luciferase (Luc) enzyme

1. 注意事项：
a） D-荧光素盐(钠盐或钾盐)在水性缓冲液（pH 6.1-6.5）中表现出高达100mM的高溶解度。储备溶液可以用不含ATP的水制备，并在-20°C的避光/黑暗中储存。游离酸必须用适当的碱中和才能溶解。在较高的pH值下，荧光素在碱性催化下会形成脱氢荧光素，并外消旋为L-异构体（L-荧光素）。
b） D-荧光素可用于任何现有的报告分析或ATP分析系统。
c） 如果测试ATP，请戴手套并使用无ATP容器，以尽量减少所有可能的ATP污染源。仅使用无菌无ATP水和试剂。使用高压灭菌水制备所有试剂。
2.实验方案：该方案仅供参考，可根据您的具体要求进行调整
以下方案是D-Luciferin钠/钾盐的制备示例。它适用于大多数细胞类型和体内动物使用。
2.1体外生物发光图像分析示例
a） 在无菌水中制备100 mM（100-200X）荧光素储备溶液。混合均匀。立即使用或一次性分装，在-20°C下避光储存，避免冻融循环。
b） 在预热的组织培养基中制备0.5-1mM D-荧光素工作溶液。
c） 从培养的细胞中吸出培养基。
d） 向细胞中加入荧光素工作溶液，在成像前在37°C下孵育5-10分钟。
2.2体内生物发光图像分析示例
a） 在DPBS中制备15mg/mL荧光素储备溶液，不含Mg2+和Ca2+。混合均匀。
b） 过滤器通过0.2μM过滤器从溶液中去除细菌。立即使用或一次性分装，并在-20°C下避光储存，以避免冻融循环。
c） 成像前10-15分钟，腹腔注射150mg/kg（或10μL/g荧光素储备溶液）动物体重的荧光素。
注意：应在每种动物模型上进行荧光素动力学研究，以确定峰值信号时间。
2.3荧光素报告基因检测示例
a） 在无菌水中制备100 mM荧光素储备溶液。立即使用或一次性分装，在-20°C下避光储存，避免冻融循环。
b） 在pH 7.8的25 mM Tricine缓冲液中制备1 mM D-荧光素工作溶液和3 mM ATP、1 mM DTT和15 mM MgSO4。
c） 将5-10μL细胞裂解液转移到微孔板上。使用裂解试剂或不含裂解物的缓冲溶液作为空白。
d） 根据制造商的说明，向发光计中注入荧光素工作溶液。
e） 立即注入200μL荧光素工作溶液，积分时间为10秒。

目前最流行的方法是生物发光（BLI），它使用D-荧光素底物和萤火虫荧光素酶（Fluc）作为报告基因。通过绘制总体信号强度与 D-荧光素注射后的时间量的关系图来创建时间-强度曲线。除了峰值信号之外，峰值信号的替代信号被识别为注射 D-荧光素后预定时间间隔（5、10、15 和 20 分钟）的信号。为了描述 D-荧光素注射后时间变化的模式，给定时间-强度曲线中的信号根据曲线中的峰值信号进行标准化 [3]。每克体重使用 10 μL D-荧光素原液（腹膜内或静脉注射）。由于常规剂量为 150 mg/kg，20 g 注射液通常应含有 200 μL。将 D-荧光素（钾盐或钠盐）溶液溶解至终浓度 15 mg/mL，将其解冻并在 dPBS（透明钙盐或镁盐）中稀释。用 5–10 mL 无菌 H2O 润湿 0.22 µM 过滤器，然后沥干。 ..将 D-荧光素溶液通过已生产的 0.22 µM 注射器过滤器。

D-荧光素是所有萤光素酶的天然底物，这些萤光素酶催化生物发光昆虫产生光。本综述涵盖了D-荧光素和衍生物或类似物的合成，它们是美国萤火虫Photinus pyralis荧光素酶的底物或抑制剂，Photinus piralis是体外和光学成像技术中更常用的酶[1]。

The peak signal or the signal at a predetermined, fixed time point after D-luciferin injection may be used for the quantitative analysis of in vivo bioluminescence imaging. We repeatedly performed sequential bioluminescence imaging after subcutaneous injection of D-luciferin in mice bearing subcutaneous tumors. The peak time in each measurement became shorter early after cell inoculation, presumably due to gradual establishment of intratumoral vasculature, and reached a plateau of about 10 min on day 10. Although the correlation between the signal at a fixed time point and the peak signal was high, the signal at 5 or 10 min normalized for the peak signal was lower for earlier days, which caused overestimation of tumor growth. The time course of the signals after D-luciferin injection may vary with time after cell inoculation, and this variation should be considered when determining the imaging protocol for quantitative bioluminescence tumor monitoring.[2]

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

[2]. Luciferin derivatives for enhanced in vitro and in vivo bioluminescence assays. Biochemistry. 2006 Sep 19;45(37):11103-12.

[3]. Timing of imaging after d-luciferin injection affects the longitudinal assessment of tumor growthusing in vivo bioluminescence imaging. Int J Biomed Imaging. 2010;2010:471408.

The fascinating phenomenon of bioluminescence and the wide range of biotechnological applications in optical imaging makes the chemistry of D-luciferin and related analogues extremely interesting. This goes beyond the early expectations originally suggested by White et al. in 1971: “it is clear that chemically produced excited states are of central importance in bioluminescence; it seems moderately certain that they will also be found to be important in other areas of biology”. 1 The synthetic approach to the preparation of the related benzothiazole system has not moved too far from the original works dating back to nearly forty years ago. However, the chemistry involved is sound and still used for the preparation of D-luciferin and related compounds. These can be very useful as substrates for luciferases in applications such as optical imaging techniques, nowadays widely used for preclinical molecular imaging in cells and small animals. The yellow-green light emitted by the classical enzyme used for this purpose, PpyLuc, is characterized by a broad emission spectrum with a maximum at 560 nm and a low background bioluminescence. This makes in vivo bioluminescence imaging with PpyLuc an easy and highly sensitive method for small-animal molecular imaging. Modulation of the wavelength of the emitted light is an important target, that has mainly been achieved by site-directed mutagenesis of amino acid residues of luciferase. The same result could perhaps be obtained by chemical manipulation of D-luciferin structure, but not many transformations are possible with a relatively simple structure such as that of D-luciferin. In fact, only a few compounds have been prepared and for a reduced number of them it has been demonstrated that the light can be shifted to red in PpyLuc-catalyzed bioluminescence. However, the examples presented in this review suggest that, in addition to biochemical engineering of the enzyme, ingenuity and fantasy of synthetic organic chemistry can bring new exciting advancement in this fascinating area of research. Finally, other analogues of D-luciferin may meet expectations for new applications of in vivo optical imaging, as recently disclosed by reports that enlighten the possibility of binding a wide variety of compounds, including peptides or functionalized PEGs, to the amino group of D-6′-aminoluciferin. [1]

---

Staining Example 1:
D-Luciferin sodium may be used as a substrate of luciferases for in vivo imaging.
Method: For bioluminescence imaging.
1). Anesthetize mice, then inject mice with D-Luciferin sodium (75 mg/kg) for image.
2). Use the bioluminescence imaging system for image.

---

Staining Example 2:
D-Luciferin sodium may be used as a substrate of luciferases for in vivo imaging to monitor tumor growth.
Method: For bioluminescence imaging.
1). Inject D-Luciferin sodium (150 mg/kg; intraperitoneal injection) into the mice.
2). Use a bioluminescence imaging system for image.

---

Staining Example 3:
D-Luciferin sodium may be used as a substrate of luciferases for a split-luciferase (LUC) assay.
Method: For split-luciferase (LUC) assay.
1). Incubate plant sample (leaves) with D-Luciferin sodium (1 mM; 10 min) luciferin.
2). Use a Photek camera to capture signals and images.

---

Staining Example 4:
D-Luciferin sodium may be used as a substrate of luciferases for in vivo imaging to monitor tumor growth.
Method: For bioluminescence imaging.
1). Inject D-Luciferin sodium (150 mg/kg; intraperitoneal injection) into the mice.
2). Use IVIS Lumina XRMS Series for bioluminescence imaging.

C11H7N2NAO3S2

302.29

301.979

C, 43.71; H, 2.33; N, 9.27; Na, 7.61; O, 15.88; S, 21.21

103404-75-7

D-Luciferin;2591-17-5;D-Luciferin potassium;115144-35-9

2733762

Typically exists as Light yellow to yellow solids at room temperature

473.7ºC at 760mmHg

240.3ºC

2.78E-10mmHg at 25°C

0.049

139.15

1

7

2

19

396

1

S1C(C2=NC3C([H])=C([H])C(=C([H])C=3S2)O[H])=N[C@@]([H])(C(=O)[O-])C1([H])[H].[Na+]

BZNVUYVALNTPBG-WJCSTRGMSA-M

InChI=1S/C11H8N2O3S2.Na/c14-5-1-2-6-8(3-5)18-10(12-6)9-13-7(4-17-9)11(15)16/h1-3,7,13H,4H2,(H,15,16)/q+1/p-1/b10-9+/t7-/m1./s1

sodium (S,E)-2-(6-oxobenzo[d]thiazol-2(6H)-ylidene)thiazolidine-4-carboxylate

D-Luciferin Sodium; D-Luciferin sodium salt; Sodium (S)-2-(6-hydroxybenzo[d]thiazol-2-yl)-4,5-dihydrothiazole-4-carboxylate; D-Luciferin Sodium; D-Luciferin, Sodium Salt; D-Luciferin (sodium); C11H7N2NaO3S2; D-Luciferin sodium salt monohydrate;

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

注意： 请将本产品存放在密封且受保护的环境中（例如氮气保护），避免吸湿/受潮和光照。

Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)

H2O : ~250 mg/mL (~826.99 mM)
DMSO : ~100 mg/mL (~330.80 mM)

配方 1 中的溶解度: ≥ 2.5 mg/mL (8.27 mM) (饱和度未知) in 10% DMSO + 40% PEG300 + 5% Tween80 + 45% Saline (这些助溶剂从左到右依次添加，逐一添加), 澄清溶液。
例如，若需制备1 mL的工作液，可将100 μL 25.0 mg/mL澄清DMSO储备液加入到400 μL PEG300中，混匀；然后向上述溶液中加入50 μL Tween-80，混匀；加入450 μL生理盐水定容至1 mL。
*生理盐水的制备：将 0.9 g 氯化钠溶解在 100 mL ddH₂O中，得到澄清溶液。

---

配方 2 中的溶解度: ≥ 2.5 mg/mL (8.27 mM) (饱和度未知) in 10% DMSO + 90% (20% SBE-β-CD in Saline) (这些助溶剂从左到右依次添加，逐一添加), 澄清溶液。
例如，若需制备1 mL的工作液，可将 100 μL 25.0 mg/mL澄清DMSO储备液加入900 μL 20% SBE-β-CD生理盐水溶液中，混匀。
*20% SBE-β-CD 生理盐水溶液的制备（4°C，1 周）：将 2 g SBE-β-CD 溶解于 10 mL 生理盐水中，得到澄清溶液。

---

View More

配方 3 中的溶解度: 100 mg/mL (330.80 mM) in PBS (这些助溶剂从左到右依次添加，逐一添加), 澄清溶液; 超声助溶.

---

请根据您的实验动物和给药方式选择适当的溶解配方/方案：
1、请先配制澄清的储备液(如：用DMSO配置50 或 100 mg/mL母液(储备液));
2、取适量母液，按从左到右的顺序依次添加助溶剂，澄清后再加入下一助溶剂。以 下列配方为例说明 (注意此配方只用于说明，并不一定代表此产品 的实际溶解配方):
10% DMSO → 40% PEG300 → 5% Tween-80 → 45% ddH2O (或 saline);
假设最终工作液的体积为 1 mL, 浓度为5 mg/mL: 取 100 μL 50 mg/mL 的澄清 DMSO 储备液加到 400 μL PEG300 中，混合均匀/澄清；向上述体系中加入50 μL Tween-80，混合均匀/澄清；然后继续加入450 μL ddH2O (或 saline)定容至 1 mL；
3、溶剂前显示的百分比是指该溶剂在最终溶液/工作液中的体积所占比例;
4、 如产品在配制过程中出现沉淀/析出，可通过加热(≤50℃)或超声的方式助溶;
5、为保证最佳实验结果，工作液请现配现用！
6、如不确定怎么将母液配置成体内动物实验的工作液，请查看说明书或联系我们;
7、 以上所有助溶剂都可在 Invivochem.cn网站购买。