L-012

L-012 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]. In Vitro: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, 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.
In Vivo: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.