Monochlorobimane

• CAT Number: I013073
• CAS Number: 76421-73-3
• Molecular Formula: C10H11ClN2O2
• Molecular Weight: 226.66
• Purity: ≥95%
• Synonyms: 7-(chloromethyl)-1,2,6-trimethylpyrazolo[1,2-a]pyrazole-3,5-dione
• InChI: InChI=1S/C10H11ClN2O2/c1-5-7(3)12-8(4-11)6(2)10(15)13(12)9(5)14/h4H2,1-3H3
• InChIKey: SUIPVTCEECPFIB-UHFFFAOYSA-N
• SMILES: CC1=C(N2C(=C(C(=O)N2C1=O)C)CCl)C

Monochlorobimane (Chlorobimane) is a fluorescent dye (λex=380 nm, λem=470 nm) to measure glutathione (GSH) in cellular assays[1].
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 H2O2)
2. Incubate the plate overnight at 37?°C, 5?% CO2. 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 EX/EM= 380/465 ±20 nm respectively (blue).

Reference

[1]. Kamencic H, et al. Monochlorobimane fluorometric method to measure tissue glutathione. Anal Biochem. 2000 Nov 1;286(1):35-7.
 [Content Brief]

[2]. Manuela D. Machado, et al. Assessment of cellular reduced glutathione content in Pseudokirchneriella subcapitata using monochlorobimane. Journal of Applied Phycology volume 24, pages1509–1516(2012).