D-luciferin is the natural substrate of the enzyme luciferase (Luc) that catalyzes the production of the typical yellowgreenlightof fireflies. The 560 nm chemiluminescence from this reaction peaks within seconds, withlightoutput 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^[1]. 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) .

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℃ 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℃ 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℃ 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 Mg²⁺和 Ca²⁺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℃ 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℃ 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。

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^[3].
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.

318.41

Solid

C₁₁H₇KN₂O₃S₂

115144-35-9

D-萤光素钾盐

Room temperature in continental US; may vary elsewhere.

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)

H₂O : 25 mg/mL(78.52 mM;Need ultrasonic)

请根据产品在不同溶剂中的溶解度选择合适的溶剂配制储备液；一旦配成溶液，请分装保存，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℃, 6 months; -20℃, 1 month (sealed storage, away from moisture and light)。-80℃ 储存时，请在 6 个月内使用，-20℃ 储存时，请在 1 个月内使用。

请根据您的实验动物和给药方式选择适当的溶解方案。以下溶解方案都请先按照In Vitro方式配制澄清的储备液，再依次添加助溶剂：

——为保证实验结果的可靠性，澄清的储备液可以根据储存条件，适当保存；体内实验的工作液，建议您现用现配，当天使用； 以下溶剂前显示的百
分比是指该溶剂在您配制终溶液中的体积占比；如在配制过程中出现沉淀、析出现象，可以通过加热和/或超声的方式助溶

• 1.

  请依序添加每种溶剂： PBS

  Solubility: 8.33 mg/mL (26.16 mM); Clear solution; Need ultrasonic and warming and heat to 60℃

• 
  Description: D-Luciferin potassium 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 potassium (75 mg/kg) for image.
  2. Use the bioluminescence imaging system for image.
• 
  Description: D-Luciferin potassium can be used as a substrate of luciferases for in vivo imaging to monitor tumor growth.
  Method: For bioluminescence imaging.
  1. Inject D-Luciferin potassium (150 mg/kg; intraperitoneal injection) into the mice.
  2. Use a bioluminescence imaging system for image.
• 
  Description: D-Luciferin potassium 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 potassium (1 mM; 10 min) luciferin.
  2. Use a Photek camera (HRPCS5, Photek) to capture signals and images.
• 
  Description: D-Luciferin can be used as a substrate of luciferases for in vivo imaging to monitor tumor growth.
  Method: For bioluminescence imaging.
  1. Inject D-Luciferin potassium (150 mg/kg; intraperitoneal injection) into the mice.
  2. Use IVIS Lumina XRMS Series (PerkinElmer, Waltham, MA) for bioluminescence imaging.