Real Thiol is a reversible reaction-based fluorescent probe which can quantitatively monitor the real-time glutathione dynamics in living cells.

Real Thiol (RT) shows ratiometric fluorescence responses with a wide dynamic range when reacting with Glutathione (GSH). Real Thiol and its GSH adduct (RT-GSH) shows fluorescence maxima at 487 and 562 nm with excitation wavelengths at 405 and 488 nm, respectively. The K_d for the reaction between Real Thiol and GSH is 3.7 mM. Real Thiol has much improved quantum yields and photostability. The wide dynamic range of Real Thiol allows for monitoring of the GSH level changes within 1 to 10 mM in both directions in living cells. Gel permeation chromatography (GPC) analysis showes that 10% of Real Thiol reacts with thiolated proteins while 90% of Real Thiol reacts with GSH^[1].
Guidelines (Following is our recommended protocol. This protocol only provides a guideline, and should be modified according to your specific needs)^[1].
1. Real Thiol (RT) is dissolved in DMSO to obtain a 10 mM stock solutions and further diluted before use.
2. Cells are cultured in 10% FBS DMEM containing different concentrations of Buthionine sulfoximine (0.9, 1.9, 3.9, 7.8, 15.6, 31.3, 62.5, 125, 250 and 500 μM) for 72 h and harvested for analysis.
3. Cells are then suspended in fresh medium containing 1 μM of Real Thiol probe as single-cell suspensions, and keep the staining time between 9 and 10 min for each sample.
4. Analyze sample on a flow cytometer. PE and pacific-blue channels were selected to measure the fluorescence intensities of unreacted and reacted Real Thiol, respectively.

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

411.36

C₂₀H₁₇N₃O₇

2280796-90-7

Room temperature in continental US; may vary elsewhere.

DMSO : ≥ 101 mg/mL (245.53 mM)

* “≥” means soluble, but saturation unknown.

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。-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。

• [1]. Jiang X, et al. Quantitative real-time imaging of glutathione. Nat Commun. 2017 Jul 13;8:16087.

Real Thiol (RT) stock solution is diluted with PBS to the desired concentrations. Equal volumes (typically 20 to 100 μL) of various Glutathione (GSH) solutions with different concentrations are mixed with the Real Thiol solution 10 to 15 min before measurement. All samples are prepared on one 96-well/384-well plate with 3 replicates each. The plate reader is set to read absorption of all samples first. Fluorescent signals are then recorded at λ_ex=405 nm, λ_em=485 nm, and λ_ex=488 nm, λ_em=565 nm with bottom read. For testing fluorescent interference by environmental factors, the Real Thiol stock solution is directly diluted with corresponding solutions (for example, certain pH buffer or glycerol solution) and measured using the plate reader with the same settings stated above^[1].

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

• [1]. Jiang X, et al. Quantitative real-time imaging of glutathione. Nat Commun. 2017 Jul 13;8:16087.

Real Thiol is a reversible reaction-based fluorescent probe which can quantitatively monitor the real-time glutathione dynamics in living cells.

Real Thiol (RT) shows ratiometric fluorescence responses with a wide dynamic range when reacting with Glutathione (GSH). Real Thiol and its GSH adduct (RT-GSH) shows fluorescence maxima at 487 and 562 nm with excitation wavelengths at 405 and 488 nm, respectively. The K_d for the reaction between Real Thiol and GSH is 3.7 mM. Real Thiol has much improved quantum yields and photostability. The wide dynamic range of Real Thiol allows for monitoring of the GSH level changes within 1 to 10 mM in both directions in living cells. Gel permeation chromatography (GPC) analysis showes that 10% of Real Thiol reacts with thiolated proteins while 90% of Real Thiol reacts with GSH^[1].
Guidelines (Following is our recommended protocol. This protocol only provides a guideline, and should be modified according to your specific needs)^[1].
1. Real Thiol (RT) is dissolved in DMSO to obtain a 10 mM stock solutions and further diluted before use.
2. Cells are cultured in 10% FBS DMEM containing different concentrations of Buthionine sulfoximine (0.9, 1.9, 3.9, 7.8, 15.6, 31.3, 62.5, 125, 250 and 500 μM) for 72 h and harvested for analysis.
3. Cells are then suspended in fresh medium containing 1 μM of Real Thiol probe as single-cell suspensions, and keep the staining time between 9 and 10 min for each sample.
4. Analyze sample on a flow cytometer. PE and pacific-blue channels were selected to measure the fluorescence intensities of unreacted and reacted Real Thiol, respectively.

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

411.36

C₂₀H₁₇N₃O₇

2280796-90-7

Room temperature in continental US; may vary elsewhere.

DMSO : ≥ 101 mg/mL (245.53 mM)

* “≥” means soluble, but saturation unknown.

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。-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。

• [1]. Jiang X, et al. Quantitative real-time imaging of glutathione. Nat Commun. 2017 Jul 13;8:16087.

Real Thiol (RT) stock solution is diluted with PBS to the desired concentrations. Equal volumes (typically 20 to 100 μL) of various Glutathione (GSH) solutions with different concentrations are mixed with the Real Thiol solution 10 to 15 min before measurement. All samples are prepared on one 96-well/384-well plate with 3 replicates each. The plate reader is set to read absorption of all samples first. Fluorescent signals are then recorded at λ_ex=405 nm, λ_em=485 nm, and λ_ex=488 nm, λ_em=565 nm with bottom read. For testing fluorescent interference by environmental factors, the Real Thiol stock solution is directly diluted with corresponding solutions (for example, certain pH buffer or glycerol solution) and measured using the plate reader with the same settings stated above^[1].

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

• [1]. Jiang X, et al. Quantitative real-time imaging of glutathione. Nat Commun. 2017 Jul 13;8:16087.