单氯二胺

Guidelines (Following is our recommended protocol. This protocol only provides a guideline, and should be modified according to your specific needs).
1. Preparation of control and experimental wells:
The experiment should consist of parallel negative, positive and experimental wells respectively.
Experimental wells: add 200 μL of cell culture media containing your GSH effector of interest at desired concentration (e.g. 200 µM H ₂ O ₂ )
2. Incubate the plate overnight at 37 °C, 5 % CO ₂ . The incubation time varies depended on your normal protocol
3. Monochlorobimane (mBCl) is added to the cells at a final concentration of 20-100 μM from a working solution of 1 mM. The stock solution of mBCl (50 mM) was prepared in dimethyl sulphoxide (DMSO) and stored at -20°C; the working solution was prepared before use by diluting the stock solution in 0.1 M PBS buffer (pH 7.0). In the assay the final concentration of DMSO was below 0.2% (v/v)
4. The cell suspensions were incubated with mBCl in the dark at 25°C for ~2 h
5. Remove the dye and treatment by centrifugation at 700 X g for 5 min. Add 200 µL of the Assay Buffer to each well and continue to the preferred method of detection
6. Detection of intracellular GSH:
Fluorescence plate reader: For adherent cells; read fluorescence directly off the culturing plate. If working with suspension cells; aliquot 200 µL of the culture suspension into the white opaque plate and record fluorescence at E _X /E _M = 380/465 ±20 nm respectively ( blue ).