HKGreen-4I is a highly sensitive green fluorescent probe that specifically detects ONOO- in living cells. Its maximum excitation wavelength and emission wavelength are 520 nm and 543 nm respectively.

Physicochemical Properties

Molecular Formula	C36H30N2O11
Molecular Weight	666.630210399628
Exact Mass	666.184
CAS #	1448821-82-6
PubChem CID	89717569
Appearance	White to pink solid powder
LogP	4.5
Hydrogen Bond Donor Count	1
Hydrogen Bond Acceptor Count	12
Rotatable Bond Count	11
Heavy Atom Count	49
Complexity	1250
Defined Atom Stereocenter Count	0
SMILES	O1C(C2C=C(C(N(CC(=O)OC)CC(=O)OC)=O)C=CC=2C21C1C=CC(=CC=1OC1C=C(C=CC2=1)N(C)C1C=CC(=CC=1)O)OC(C)=O)=O
InChi Key	ZOZXUNDNNSKANR-UHFFFAOYSA-N
InChi Code	InChI=1S/C36H30N2O11/c1-20(39)47-25-11-14-29-31(17-25)48-30-16-23(37(2)22-6-9-24(40)10-7-22)8-13-28(30)36(29)27-12-5-21(15-26(27)35(44)49-36)34(43)38(18-32(41)45-3)19-33(42)46-4/h5-17,40H,18-19H2,1-4H3
Chemical Name	methyl 2-[[3'-acetyloxy-6'-(4-hydroxy-N-methylanilino)-3-oxospiro[2-benzofuran-1,9'-xanthene]-5-carbonyl]-(2-methoxy-2-oxoethyl)amino]acetate
HS Tariff Code	2934.99.9001
Storage	Powder-20°C 3 years 4°C 2 years In solvent -80°C 6 months -20°C 1 month Note: This product requires protection from light (avoid light exposure) during transportation and storage.
Shipping Condition	Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)

Biological Activity

ln Vitro

1. Preparation of HKGreen-4I working solution 1.1 Preparation of stock solution: Dilute 1 mg of HKGreen-4I with 150 μL of anhydrous DMSO to prepare a 10 mM stock solution. It is advised to aliquot and store HKGreen-4I storage solution at -20°C or -80°C in the dark. 1.2 Creating a functional solution Use preheated serum-free cell culture medium or PBS dilution stock solution to prepare a 1-10 μM HKGreen-4I working solution. Note: Please prepare the HKGreen-4I working fluid for usage by adjusting its concentration to suit the current circumstances. 2. Cell staining (suspension cells) 2.1 Centrifuge the cells, add PBS, and wash twice for five minutes each time. The density of cells is 1×106/mL. 2.2 Add 1 mL of the HKGreen-4I working solution, then let it sit at room temperature for a duration of 5 to 30 minutes. 2.3 Centrifuge for 3–4 minutes at 400 g, then remove the supernatant. 2.4 After adding PBS, wash the cells twice for five minutes each. 2.5 Re-suspend the cells in 1 milliliter of PBS or serum-free media, and use a flow cytometer or fluorescence microscope to observe. 3. Staining adherent cells in cells 3.1 Use sterile coverslips to cultivate adherent cells. 3.2 Aspirate extra culture medium after removing the coverslip from the medium. 3.3 Cover the cells fully with 100 μL of the dye working solution, shake gently, and allow incubate for five to thirty minutes. 3.4 Use a flow cytometer or fluorescence microscope to observe after aspirating the dye working solution and washing it twice in culture media for a total of five minutes each time.

References

[1]. Trends Cell Biol. 2007 Sep;17(9):422-7. doi: 10.1016/j.tcb.2007.07.009. Epub 2007 Sep 4. [2]. Diarylamine-based fluorogenic probes for detection of peroxynitrite. US9651528B2. [3]. Tandem Payne/Dakin Reaction: A New Strategy for Hydrogen Peroxide Detection and Molecular Imaging. Angew Chem Int Ed Engl. 2018 Aug 6;57(32):10173-10177.

Solubility Data

Solubility (In Vitro)

DMSO: 40 mg/mL (60.00 mM)

Solubility (In Vivo)

Solubility in Formulation 1: ≥ 6 mg/mL (9.00 mM) (saturation unknown) in 10% DMSO + 40% PEG300 + 5% Tween80 + 45% Saline (add these co-solvents sequentially from left to right, and one by one), clear solution. For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 60.0 mg/mL clear DMSO stock solution to 400 μL PEG300 and mix evenly; then add 50 μL Tween-80 to the above solution and mix evenly; then add 450 μL normal saline to adjust the volume to 1 mL. Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH₂ O to obtain a clear solution. Solubility in Formulation 2: 3 mg/mL (4.50 mM) in 10% DMSO + 90% (20% SBE-β-CD in Saline) (add these co-solvents sequentially from left to right, and one by one), suspension solution; with ultrasonication. For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 30.0 mg/mL clear DMSO stock solution to 900 μL of 20% SBE-β-CD physiological saline solution and mix evenly. Preparation of 20% SBE-β-CD in Saline (4°C，1 week): Dissolve 2 g SBE-β-CD in 10 mL saline to obtain a clear solution. Solubility in Formulation 3: ≥ 1.75 mg/mL (2.63 mM) (saturation unknown) in 10% DMSO + 90% Corn Oil (add these co-solvents sequentially from left to right, and one by one), clear solution. For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 17.5 mg/mL clear DMSO stock solution to 900 μL of corn oil and mix evenly.  (Please use freshly prepared in vivo formulations for optimal results.)

Preparing Stock Solutions		1 mg	5 mg	10 mg
	1 mM	1.5001 mL	7.5004 mL	15.0008 mL
	5 mM	0.3000 mL	1.5001 mL	3.0002 mL
	10 mM	0.1500 mL	0.7500 mL	1.5001 mL

*Note: Please select an appropriate solvent for the preparation of stock solution based on your experiment needs. For most products, DMSO can be used for preparing stock solutions (e.g. 5 mM, 10 mM, or 20 mM concentration); some products with high aqueous solubility may be dissolved in water directly. Solubility information is available at the above Solubility Data section. Once the stock solution is prepared, aliquot it to routine usage volumes and store at -20°C or -80°C. Avoid repeated freeze and thaw cycles.