5(6)-Carboxy-2',7'-dichlorofluorescein diacetate 

5 (6)-Carboxy-2',7'-dichlorofluorescein diacetate is a fluorescein-based reactive oxygen species (ROS) probe and also a MRP2 substrate. 5 (6)-Carboxy-2',7'-dichlorofluorescein diacetate serves as a substrate for intracellular esterases, which cleave its acetate groups to generate a fluorescent product capable of detecting intracellular ROS. 5 (6)-Carboxy-2',7'-dichlorofluorescein diacetate is ATP-dependent and is transported via a single MRP2 binding site; it competes with LTC₄ for MRP2 binding sites and inhibits MRP2-mediated LTC₄ transport (Ex/Em = 496/525 nm)^[1][2][3][4].

Guide (The following is our recommended protocol. This protocol is only a guide and should be modified according to your specific needs).
1. Dye preparation
1.1 Preparation of stock solution
Use anhydrous DMSO to prepare a 10 mM stock solution.
Note: It is recommended that the stock solution be stored at -20°C or -80°C in the dark after aliquoting.
1.2 Preparation of working solution
Use preheated serum-free cell culture medium or PBS to dilute the stock solution to prepare a 1-10 μM working solution.
Note: Please adjust the concentration of H2DCFDA working solution according to actual conditions and prepare it before use.
2. Cell staining (suspended cells)
2.1 Collect cells by centrifugation and wash twice with PBS for 5 minutes each time. The cell density is 1×10⁶/mL.
2.2 Add dye working solution and incubate at room temperature for 5-30 minutes.
2.3 Centrifuge at 400 g for 3-4 minutes and discard the supernatant.
2.4 Add PBS to wash the cells twice, 5 minutes each time.
2.5 Resuspend the cells in of serum-free medium or PBS and observe using a fluorescence microscope or flow cytometer.
3. Cell staining (adherent cells)
3.1 Culture the adherent cells on a sterile coverslip.
3.2 Remove the coverslip from the culture medium and remove the excess culture medium.
3.3 Add of dye working solution, gently shake to completely cover the cells, and incubate for 5-30 minutes.
3.4 Aspirate the dye working solution, wash 2-3 times with culture medium, 5 minutes each time, and observe using a fluorescence microscope or flow cytometer.
Note: If flow cytometry is required, the cells need to be digested with trypsin and resuspended before staining.

5(6)-Carboxy-2',7'-dichlorofluorescein diacetate (sufficient to determine Michaelis-Menten kinetics; up to 30 min) acts as an active, single-binding-site substrate of MRP2 in Sf9 cell membrane vesicles, with a K_m of 12.3 μM, and functions as a valid fluorescent surrogate for LTC4 in MRP2 vesicular transport assays^[1].
5(6)-Carboxy-2',7'-dichlorofluorescein diacetate (10 μM) is a highly sensitive, long-lasting cell-permeant tracer that detects ROS generation in primary human neuronal-glial (HNG) cell co-cultures, with an intracellular fluorescence persistence at least 10-fold longer than non-carboxylated 2′,7′-dichlorofluorescein diacetate^[2].

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

529.28

C₂₅H₁₄Cl₂O₉

127770-45-0

Solid

Off-white to light yellow

525

496

ClC1=C(OC(C)=O)C=C2OC3=CC(OC(C)=O)=C(Cl)C=C3C(OC4=O)(C5=CC=C(C(O)=O)C=C54)C2=C1.ClC6=C(OC(C)=O)C=C7OC8=CC(OC(C)=O)=C(Cl)C=C8C(OC9=O)(C%10=CC(C(O)=O)=CC=C%109)C7=C6

Room temperature in continental US; may vary elsewhere.

* Please refer to the solubility information to select the appropriate solvent. Once prepared, please aliquot and store the solution to prevent product inactivation from repeated freeze-thaw cycles.
Storage method and period of stock solution: -80°C, 6 months; -20°C, 1 month. When stored at -80°C, please use it within 6 months. When stored at -20°C, please use it within 1 month.

• [1]. Heredi-Szabo K, et al. Characterization of 5(6)-carboxy-2,'7'-dichlorofluorescein transport by MRP2 and utilization of this substrate as a fluorescent surrogate for LTC4. J Biomol Screen. 2008;13(4):295-301.  [Content Brief]

  [2]. Pogue AI, et al. Metal-sulfate induced generation of ROS in human brain cells: detection using an isomeric mixture of 5- and 6-carboxy-2',7'-dichlorofluorescein diacetate (carboxy-DCFDA) as a cell permeant tracer. Int J Mol Sci. 2012;13(8):9615-9626.  [Content Brief]

  [3]. Munić V, et al. Characterization of rhodamine-123, calcein and 5(6)-carboxy-2',7'-dichlorofluorescein (CDCF) export via MRP2 (ABCC2) in MES-SA and A549 cells. Eur J Pharm Sci. 2011;43(5):359-369.  [Content Brief]

  [4]. Zamek-Gliszczynski MJ, et al. Pharmacokinetics of 5 (and 6)-carboxy-2',7'-dichlorofluorescein and its diacetate promoiety in the liver. J Pharmacol Exp Ther. 2003;304(2):801-809.  [Content Brief]