Monochlorobimane (Chlorobimane) is a fluorescent dye (λ_ex=380 nm, λ_em=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 E_X/E_M= 380/465 ±20 nm respectively (blue).

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

226.66

C₁₀H₁₁ClN₂O₂

76421-73-3

Room temperature in continental US; may vary elsewhere.

-20deg;C, protect from light

*In solvent : -80deg;C, 6 months; -20deg;C, 1 month (protect from light)

In Vitro:;

DMSO : 50 mg/mL (220.59 mM; Need ultrasonic)

*

请根据产品在不同溶剂中的溶解度选择合适的溶剂配制储备液；一旦配成溶液，请分装保存，避免反复冻融造成的产品失效。
储备液的保存方式和期限：-80°C, 6 months; -20°C, 1 month (protect from light)。-80°C 储存时，请在 6 个月内使用，-20°C 储存时，请在 1 个月内使用。

In Vivo:

请根据您的实验动物和给药方式选择适当的溶解方案。以下溶解方案都请先按照 In Vitro 方式配制澄清的储备液，再依次添加助溶剂：

——为保证实验结果的可靠性，澄清的储备液可以根据储存条件，适当保存；体内实验的工作液，建议您现用现配，当天使用； 以下溶剂前显示的百
分比是指该溶剂在您配制终溶液中的体积占比；如在配制过程中出现沉淀、析出现象，可以通过加热和/或超声的方式助溶

• 1.

  请依序添加每种溶剂：;10% DMSO ;; 40% PEG300 ;; 5% Tween-80 ;; 45% saline

  Solubility: 2.5 mg/mL (11.03 mM); Clear solution; Need ultrasonic

  此方案可获得 2.5 mg/mL (11.03 mM) 的澄清溶液。

  以 1 mL 工作液为例，取 100 μL 25.0 mg/mL 的澄清 DMSO 储备液加到 400 μL PEG300 中，混合均匀；向上述体系中加入50 μL Tween-80，混合均匀；然后继续加入 450 μL生理盐水定容至 1 mL。

  将 0.9 g 氯化钠，完全溶解于 100 mL ddH₂O 中，得到澄清透明的生理盐水溶液

• 2.

  请依序添加每种溶剂：;10% DMSO ;; 90% (20% SBE-β-CD in saline)

  Solubility: 2.5 mg/mL (11.03 mM); Suspended solution; Need ultrasonic

  此方案可获得 2.5 mg/mL (11.03 mM) 的均匀悬浊液，悬浊液可用于口服和腹腔注射。

  以 1 mL 工作液为例，取 100 μL 25.0 mg/mL 的澄清 DMSO 储备液加到 900 μL 20% 的 SBE-β-CD 生理盐水水溶液中，混合均匀。

  将 2 g 磺丁基醚 β-环糊精加入 5 mL 生理盐水中，再用生理盐水定容至 10 mL，完全溶解，澄清透明

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

  [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).

Monochlorobimane (mCB) is added to the second half liver tissue homogenate to a final concentration of 100 mM along with glutathione S-transferase (1 U/mL) obtained from equine liver; the homogenate is then allowed to incubate at room temperature for 30 min. The Glutathione (GSH)-Monochlorobimane adduct is measured in a microtiter reader^[1].

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

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

  [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).