5-IAF is an idoacetamide derivate of fluoresceine.

8 µM GSH sample solution is derivatized with 8, 80, 200, 400, 800, 1600, 2000, 2500 µM 5-IAF, respectively and analyzed with CE-LIF. The highest signal intensity is obtained with 400 and 800 µM 5-IAF^[2].

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

515.25

C₂₂H₁₄INO₆

63368-54-7

5-碘乙酰氨基荧光素

Room temperature in continental US; may vary elsewhere.

Powder	-20deg;C	3 years
In solvent	-80deg;C	6 months
	-20deg;C	1 month

In Vitro:;

DMSO : 50 mg/mL (97.04 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。-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: 1.43 mg/mL (2.78 mM); Suspended solution; Need ultrasonic

  此方案可获得 1.43 mg/mL (2.78 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 14.3 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: 1.43 mg/mL (2.78 mM); Suspended solution; Need ultrasonic

  此方案可获得 1.43 mg/mL (2.78 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 14.3 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]. Salvatore Sotgia, et al. Plasma L-Ergothioneine Measurement by High-Performance Liquid Chromatography and Capillary Electrophoresis after a Pre-Column Derivatization with 5-Iodoacetamidofluorescein (5-IAF) and Fluorescence Detection. PLoS One. 2013; 8(7): e70374.

  [2]. Yan Wang, et al. Determination of free and protein-bound glutathione in HepG2 cells using capillary electrophoresis with laser-induced fluorescence detection. J Chromatogr A. 2009 Apr 17;1216(16):3533-7.

HepG2 cells are lysed in 1 mL 2% acetonitrile in water, and transfered to a 1.5 mL centrifuge tube. An additional 200 µL 2% acetonitrile is used to rinse the dish and combined with the cell extract. To measure the free GSH concentration, three aliquots of 80 µL cell extract are immediately transferred to 0.5 mL centrifuge tubes after a quick mix. Then 20 µL 50 µM NAC is added, followed by the addition of 100 µL 400 µM 5-IAF to the mixture. The derivatization is conducted at room temperature in dark for 1 h, after which the mixtures are centrifuged at 9000× g for 5 min to sediment the insoluble proteins. 10 µL supernatant is diluted 100× with water and analyzed immediately by CE-LIF^[2].

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

• [1]. Salvatore Sotgia, et al. Plasma L-Ergothioneine Measurement by High-Performance Liquid Chromatography and Capillary Electrophoresis after a Pre-Column Derivatization with 5-Iodoacetamidofluorescein (5-IAF) and Fluorescence Detection. PLoS One. 2013; 8(7): e70374.

  [2]. Yan Wang, et al. Determination of free and protein-bound glutathione in HepG2 cells using capillary electrophoresis with laser-induced fluorescence detection. J Chromatogr A. 2009 Apr 17;1216(16):3533-7.