Fluo-3AM  (Synonyms: Fluo-3-pentaacetoxymethyl ester)

Fluo-3 AM is a fluorecent Ca²⁺ chelator, with high affinity for calcium. Fluo-3 AM can specifically identify intracellular calcium ions, with high sensitivity, low cytotoxicity, increased AM acetylmethyl ester can enter the cell well, after being sheared by the intracellular esterase stay in the cell to bind to calcium ions, produce strong fluorescence^[1].

Guide (The following is our recommended experimental protocol. This protocol is for reference only and should be modified according to your specific needs).
1. Preparation of Fluo-3 AM working solution
1.1 Preparation of stock solution
Dilute Fluo-3 AM with anhydrous DMSO to prepare a 10 mM stock solution.
Note: It is recommended to aliquot the Fluo-3 AM stock solution and store at -20°C or -80°C protected from light.
1.2 Preparation of working solution
Dilute the stock solution with pre-warmed HBSS to prepare a 1-10 μM Fluo-3 AM working solution.
Note: Adjust the concentration of Fluo-3 AM working solution according to actual needs and prepare fresh before use.
2. Cell staining (suspension cells)
2.1 Collect cells by centrifugation and wash twice with PBS, 5 min each time. Cell density: 1×10⁶/mL.
2.2 Add 1 mL Fluo-3 AM working solution and incubate at room temperature for 5-30 min.
2.3 Centrifuge at 400 g for 3-4 min and discard the supernatant.
2.4 Wash cells twice with PBS, 5 min each time.
2.5 Resuspend cells in 1 mL HBSS and observe using a fluorescence microscope or flow cytometer.
3. Cell staining (adherent cells)
3.1 Culture adherent cells on sterile coverslips.
3.2 Remove the coverslips from the culture medium and aspirate excess medium.
3.3 Add 100 μL dye working solution, gently shake to fully cover the cells, and incubate for 5-30 min.
3.4 Remove the dye working solution, wash 2-3 times with culture medium (5 min each), and observe using a fluorescence microscope.

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

1129.85

C₅₁H₅₀Cl₂N₂O₂₃

121714-22-5

Solid

Brown to reddish brown

526

488

ClC(C=C1C(C2=CC(OCCOC(C=C(C)C=C3)=C3N(CC(OCOC(C)=O)=O)CC(OCOC(C)=O)=O)=C(N(CC(OCOC(C)=O)=O)CC(OCOC(C)=O)=O)C=C2)=C(C=C4Cl)C5=CC4=O)=C(C=C1O5)OCOC(C)=O

Room temperature in continental US; may vary elsewhere.

-20°C, protect from light

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

* 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 (protect from light). When stored at -80°C, please use it within 6 months. When stored at -20°C, please use it within 1 month.

• [1]. Loughrey, C. M., MacEachern, K. E., Cooper, J., & Smith, G. L. (2003). Measurement of the dissociation constant of Fluo-3 for Ca2+ in isolated rabbit cardiomyocytes using Ca2+ wave characteristics. Cell Calcium, 34(1), 1–9. doi:10.1016/s0143-4160(03)00012-5  [Content Brief]