D-luciferin is the natural substrate of the enzyme luciferase (Luc) that catalyzes the production of the typical yellowgreen light of fireflies. The 560 nm chemiluminescence from this reaction peaks within seconds, with light output that is proportional to luciferase concentration when the substrate luciferin is present in excess. The luciferase (luc) gene is a popular reporter gene for research and drug screening. Chemiluminescent techniques are virtually background-free, making the luc reporter gene ideal for detecting low-level gene expression. As little as 0.02 pg of luciferase can be reliably measured in a standard scintillation counter. In addition to its role as a reporter of gene expression, luciferase is commonly used in an extremely sensitive assay for ATP. We of er the firefly luciferase (HY-P1004), luciferin free acid (HY-12591A), as well as its water-soluble sodium salts (HY-12591) and potassium salts (HY-12591B) .

Solid

1. 操作前注意事项
a) D-Luciferin 在 100 mM 的水性缓冲液 (pH 6.1-6.5) 中易于溶解。储备溶液可在不含 ATP 的水中制成，并储存在 -20°C 的温度下，以防光线照射。游离酸必须用适当的碱中和以溶解。在较高的 pH 值下，荧光素经历碱催化的脱氢脲醛生成，以及外消旋成L-异构体。
b) D-Luciferin 可用于任何现有的报告基因检测或 ATP 检测系统。
c) 如果检测 ATP，需要戴手套和使用不含 ATP 的容器，尽量减少所有可能的 ATP 污染源。只使用无菌的不含 ATP 的水和试剂。所有试剂制剂使用蒸压水。
2. 实验操作
此说明仅提供实验参考，具体实验方法根据您的具体实验进行更改。
2.1 体外成像实验示例
a) 在无菌水中制备100 mM (100-200X) 荧光素储备溶液 拌匀。立即使用或一次性等份使用，并在 -20°C 下储存，避免冻融循环，避免暴露在阳光下。
b) 在预热过的组织培养液中配制 0.5-1 mM D-Luciferin 工作液
c) 除去细胞中的培养基。
d) 在细胞中加入 D-Luciferin 工作液，成像前 37°C 孵育细胞 5-10 分钟。
2.2 体内成像实验示例
a) 在 DPBS 中制备 15 mg/mL 荧光素储备溶液，不含 Mg 和 Ca 拌匀。
b) 过滤器通过 0.2μm 过滤器对溶液进行灭菌。立即使用，或一次性等份使用，并在 -20°C 下储存，避免冻融循环，避免暴露在阳光下。
c) 成像前 10-15 分钟，以 150 mg/kg (或 10 μL/g 荧光素储备溶液) 的动物体重腹腔注射 D-Luciferin。
注：应为每个动物模型进行 D-Luciferin 动力学研究，以确定峰值信号时间。
2.3 基因检测实验示例
a) 在无菌水中制备100 mM 荧光素储备溶液。立即使用，或一次性等份使用，并在 -20°C 下储存，避免冻融循环，避免暴露在阳光下。
b) 在 25 mM tricine 缓冲液 (pH 7.8) 中制备含有 3 mM ATP、1 mM DTT 和 15 mM MgSO₄ 的 1 mM D-Luciferin 工作溶液。
c) 用移液管将 5-10 μL 细胞裂解液移到微孔板中。使用不含裂解物的裂解试剂或缓冲液作为空白对照。
d) 根据说明，使用荧光素工作溶液为光度计注入底部。
e) 立即注入 200 μL D-Luciferin工作溶液，结合时间为10秒。

Medlife has not independently confirmed the accuracy of these methods. They are for reference only.

Bioluminescence imaging (BLI) using the firefly luciferase (Fluc) as a reporter gene and D-luciferin as a substrate is currently the most widely employed technique. The total signal intensity is plotted against the time after D-luciferin injection to generate a time-intensity curve. In addition to the peak signal, the signals at fixed time points (5, 10, 15, and 20?min) after D-luciferin injection are determined as alternatives to the peak signal. The signal in a given time-intensity curve is normalized for the peak signal in the curve to represent the pattern of temporal changes after D-luciferin injection.
Inject with 10 μL of D-luciferin (intraperitoneally or intravenously) stock solution per gram of body weight: normally ~200 μL for a 20 g mouse for a standard 150 mg/kg injection.
Thaw D-Luciferin (either Potassium or Sodium Salt) at room temperature and dissolve in dPBS (no calcium or magnesium) to a final concentration of 15 mg/mL. Pre-wet a 0.22 μm filter by drawing through 5-10 mL of sterile H₂O and discard water. Sterilize the D-Luciferin solution through the prepared 0.22 μm syringe filter.

Medlife has not independently confirmed the accuracy of these methods. They are for reference only.

Room temperature or refrigerated transportation.

4°C, sealed storage, away from moisture and light

*In solvent : -80°C, 6 months; -20°C, 1 month (sealed storage, away from moisture and light)

• Description: D-Luciferin sodium can 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).
  2. Use a bioluminescence imaging system for image.
• Description: D-Luciferin sodium can 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.
• Description: D-Luciferin sodium can 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 (HRPCS5, Photek) to capture signals and images.
• Description: D-Luciferin sodium can 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 (PerkinElmer, Waltham, MA) for bioluminescence imaging.

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

  [2]. Rajesh Shinde, et al. Luciferin derivatives for enhanced 体外研究 and in vivo bioluminescence assays. Biochemistry. 2006 Sep 19;45(37):11103-12.  [Information]

  [3]. Inoue Y, et al. 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.  [Information]

产品不同，其溶解度不同。建议根据产品选择合适的溶剂配制储备溶液；配成溶液后，建议分装保存，避免反复冻融造成的产品失效。
储备液的保存方式和期限：-80°C, 6 months; -20°C, 1 month (sealed storage, away from moisture and light)。-80°C 储存时，建议在 6 个月内使用，-20°C 储存时，建议在 1 个月内使用。