L 012 sodium salt 

L 012 sodium salt is a luminal-based chemiluminescent probe. L 012 sodium salt can detect NADPH oxidase (Nox)-derived superoxide and nitrogen species (reactive oxygen species (ROS) and reactive nitrogen species (RNS))^[1][2].

Guide (The following is our recommended protocol. This protocol is only a guide and should be modified according to your specific needs).
1. Solution preparation^[1]
1.1 Preparation of stock solution
Solvent: DMSO or ultrapure water
Concentration: 10 mM is recommended.
Storage: Store at -20°C or -80°C in dark after aliquoting. Avoid repeated freezing and thawing.
1.2 Preparation of working solution
Dilute to 1-10 μM with PBS or serum-free cell culture medium (optimized according to the experiment).
Note: The working solution should be prepared and used immediately. Keep it away from light.

2. Assay for ROS Generated by Cell-Free Systems
2.1 In the presence of 1 mM hypoxanthine in KRP (Krebs-Ringer phosphate buffer), superoxide is generated by xanthine oxidase (6.4 mU/mlL) at 37°C.
2.2 Concentrations of L 012 sodium salt, luminol and MCLA used for the assay are 4 μM, 100 μM, and 4 μM, respectively.
2.3 The hydroxyl radical is generated in KRP by incubating 100 μM DTPA, 1 mM H2O2, and 50 μM FeSO4 at 37°C.
2.4 Detection using in Luminescence Reader.

MedChemExpress (MCE) has not independently confirmed the accuracy of these methods. They are for reference only.

NF-κB-luciferase reporter mice^[2].
Luciferase activity was monitored in vivo by injecting the substrate luciferin.
L-012 (25 mg/kg in 100 μL) is administered intravenously (iv), intraperitoneally (ip), or subcutaneously (sc).
Detection using in vivo imaging.
Image exposure times are between 5 s and 2 min, depending on the signal strength.

MedChemExpress (MCE) has not independently confirmed the accuracy of these methods. They are for reference only.

310.67

C₁₃H₈ClN₄NaO₂

143556-24-5

Solid

White to yellow

O=C1C2=C(C(Cl)=NC(C3=CC=CC=C3)=C2N)C(O[Na])=NN1

Room temperature in continental US; may vary elsewhere.

4°C, protect from light, stored under nitrogen

*In solvent : -80°C, 6 months; -20°C, 1 month (protect from light, stored under nitrogen)

* Please refer to the solubility information to select the appropriate solvent. Once prepared, please aliquot and store the solution to prevent product inactivation from repeated freeze-thaw cycles.
Storage method and period of stock solution: -80°C, 6 months; -20°C, 1 month (protect from light, stored under nitrogen). When stored at -80°C, please use it within 6 months. When stored at -20°C, please use it within 1 month.

* Note: If you choose water as the stock solution, please dilute it to the working solution, then filter and sterilize it with a 0.22 μm filter before use.

• [1]. Zielonka J, et al. On the use of L-012, a luminol-based chemiluminescent probe, for detecting superoxide and identifying inhibitors of NADPH oxidase: a reevaluation. Free Radic Biol Med. 2013;65:1310-1314.  [Content Brief]

  [2]. Kielland A, et al. In vivo imaging of reactive oxygen and nitrogen species in inflammation using the luminescent probe L-012. Free Radic Biol Med. 2009;47(6):760-766.  [Content Brief]