Lucigenin

Lucigenin is a chemiluminescence probe. Lucigenin can be used to detect the production of endogenous superoxide anion radical (O^2-). Lucigenin is extremely sensitive to chloride ions, while it combined with chloride ions, the fluorescence will be quenched. Lucigenin also can be used as a chloride indicator. Ex/Em=455/505 nm^[1]. In Vitro: Preparation of Lucigenin working solution
1.1 Preparation of the stock solution
Dissolve 1 mg of Lucigenin in 0.1919 mL of DMSO to obtain 10 mM of Lucigenin.
Note: It is recommended to store the stock solution at -20 °C -80 °C away from light and avoid repetitive freeze-thaw cycles.
1.2 Preparation of Lucigenin working solution
Dilute the stock solution in serum-free cell culture medium or PBS to obtain 5-10 μM of Lucigenin working solution.
Note: Please adjust the concentration of Lucigenin working solution according to the actual situation.

Cell staining
2.1 Cell preparation.
For suspension cells: Centrifuge at 1000 g at 4°C for 3-5 minutes and then discard the supernatant. Wash twice with PBS, 5 minutes each time.
For adherent cells: Discard the cell culture medium, and add trypsin to dissociate cells to make a single-cell suspension. Centrifuge at 1000 g at 4°C for 3-5 minutes and then discard the supernatant. Wash twice with PBS, 5 minutes each time.
2.2 Add 1 mL of Lucigenin working solution, and then incubate at room temperature for 15 minutes.
2.3 Centrifuge at 400 g at 4°C for 3-4 minutes and then discard the supernatant.
2.4 Wash twice with PBS, 5 minutes each time.
2.5 Resuspend cells with serum-free cell culture medium or PBS, and then detect by fluorescence microscope.